Generating regionalized neuronal cells from pluripotency, a step-by-step protocol

Abstract

Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or in vitro analysis.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

“…Thus, monolayer cultures of NSCs/NPCs would be advantageous. Several protocols to derive NSCs/NPCs efficiently from hES cells or iPSCs currently utilize Noggin to induce neuronal differentiation, 19,20 but the need for Noggin significantly increases the cost of the differentiation process.…”

Transplantation in the nonhuman primate MPTP model of Parkinson's disease: update and perspectives

“…When transplanted in the lesioned rodent striatum, DA neurons from human fVM and PSCs show extensive reinnervation of striatal and extra-striatal target structures (Brundin et al, 1986;Sanchez-Pernaute et al, 2005;Thompson et al, 2005;Kirkeby et al, 2012;Grealish et al, 2014), and, when grafted in the SN, they project axons over long distances and reinnervate the relevant A9 and A10 host target structures (Grealish et al, 2014), as was observed for rodent DA neurons (Thompson et al, 2009).…”

“…Characterization of the cells showed that DA neurons express midbrain DA neuron markers such as NURR1 and LMX1A, LMX1B, FOXA2, OTX2, CORIN, PITX3, factors that control specification and differentiation of midbrain DA neurons during mouse development (reviewed in Arenas et al, 2015), and GIRK2, which is the A9-specific marker (Thompson et al, 2005). They also express tyrosine hydroxylase (TH), and the dopamine transporter (DAT), and they produce dopamine, confirming that they are functional DA neurons (Kriks et al, 2011;Kirkeby et al, 2012;Arenas et al, 2015).…”

“…Complete and robust midbrain specification was recently obtained via a floor plate intermediate stage from human (Kriks et al, 2011;Xi et al, 2012;Sundberg et al, 2013) and NHP PSCs (Xi et al, 2012;Hallett et al, 2015;Wang et al, 2015), using a modified dual-SMAD inhibition protocol (BMP/TGFβ inhibition; Chambers et al, 2009) and activation of Wnt signalling, an essential pathway in DA neuron development in the mouse (Castelo-Branco et al, 2003Joksimovic et al, 2009) and in humans (La Manno et al, 2016). Combining suspension culture with dual-SMAD inhibition, Wnt and SHH activation also led to robust VM differentiation, with correct midbrain GIRK2+ A9 and CALBINDIN+ A10 phenotypes, similar to fVM content (Kirkeby et al, 2012;Morizane et al, 2013;Doi et al, 2014;Grealish et al, 2014;Chen et al, 2016).…”

Developing stem cell-based therapies for neural repair

“…This will require robust and appropriate fate restriction into more specialized lineages and their survival and appropriate functional integration into existing host circuitry after transplantation. Kirkeby et al (2013) contribute a detailed methodological paper on the generating regionally specified neurons, by way of controlling dorso-ventral and rostrocaudal patterning of stem cells. Arber and Li (2013) provide a more focused review of the literature on understanding cortical interneuron development and the progress in protocols to yield interneuron subpopulations from pluripotent stem cells for the purpose of transplantation into models of epilepsy.…”