A Simplified Method for Generating Purkinje Cells from Human-Induced Pluripotent Stem Cells

Abstract

The establishment of a reliable model for the study of Purkinje cells in vitro is of particular importance, given their central role in cerebellar function and pathology. Recent advances in induced pluripotent stem cell (iPSC) technology offer the opportunity to generate multiple neuronal subtypes for study in vitro. However, to date, only a handful of studies have generated Purkinje cells from human pluripotent stem cells, with most of these protocols proving challenging to reproduce. Here, we describe a simplified method for the reproducible generation of Purkinje cells from human iPSCs. After 21 days of treatment with factors selected to mimic the self-inductive properties of the isthmic organiser—insulin, fibroblast growth factor 2 (FGF2), and the transforming growth factor β (TGFβ)-receptor blocker SB431542—hiPSCs could be induced to form En1-positive cerebellar progenitors at efficiencies of up to 90%. By day 35 of differentiation, subpopulations of cells representative of the two cerebellar germinal zones, the rhombic lip (Atoh1-positive) and ventricular zone (Ptf1a-positive), could be identified, with the latter giving rise to cells positive for Purkinje cell progenitor-specific markers, including Lhx5, Kirrel2, Olig2 and Skor2. Further maturation was observed following dissociation and co-culture of these cerebellar progenitors with mouse cerebellar cells, with 10% of human cells staining positive for the Purkinje cell marker calbindin by day 70 of differentiation. This protocol, which incorporates modifications designed to enhance cell survival and maturation and improve the ease of handling, should serve to make existing models more accessible, in order to enable future advances in the field.Electronic supplementary materialThe online version of this article (10.1007/s12311-017-0913-2) contains supplementary material, which is available to authorized users.

Single-cell sequencing of human iPSC-derived cerebellar organoids shows recapitulation of cerebellar development

“…hiPSCs from a healthy 67-year-old female donor (AHO17-3) were used as previously described (Watson et al, 2018) between passage 25-35. Samples were tested and free of Mycoplasma using the MycoAlert kit (Lonza, Switzerland).…”

“…Insulin to mimic the self-inductive properties of the isthmic organizer and reproduce MHB development ( Figure 1A) (Muguruma et al, 2015;Watson et al, 2018). We refer to cellular aggregates as embryoid bodies (EBs) until the appearance of polarized neuroepithelial tissue and confirmation of MHB identity at day 21, from which point they are referred to as organoids.…”

“…Human induced pluripotent stem cells (hiPSCs) offer a powerful system to model human cerebellar development and its associated disorders. Different methodologies have recently been developed to differentiate hPSCs into cerebellar neurons through the addition of growth factors that reproduce early patterning events in vitro, recapitulating MHB barrier establishment and subsequent formation of polarized 4 cerebellar tissue characteristic of RL/VZ expansion (Ishida et al, 2016;Muguruma et al, 2015;Sundberg et al, 2018;Watson et al, 2018). Current models have mainly focussed on the differentiation of hPSC-derived Purkinje cells through co-culture with mouse cerebellar progenitors.…”

Human-Induced Pluripotent Stem Cells and Herbal Small-Molecule Drugs for Treatment of Alzheimer’s Disease

“…iPSCs are pluripotent stem cells formed by the lentiviral vector transfer of four transcription factors (i.e., Oct4, Sox2, c-Myc, and Klf4) into adult cells [19]. The benefits of iPSCs are that they differentiate into different types of neuronal cells from the stem cell stage and can be used to produce forebrain acetylcholine neurons, dopaminergic progenitor cells, and Purkinje cells [20][21][22][23]. Neuronal cells derived from iPSCs exhibit electrophysiological responses.…”

“…Damage to Purkinje cells is related to cognitive impairment and motor deficits in patients with AD [31]. The iPSC-induced increase in the number of Purkinje cell bodies is a potential novel strategy for AD treatment [22,32].…”