Cultivation, differentiation, and lentiviral transduction of human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons for studying human TAU

Abstract

TAU pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these diseases, e.g., Alzheimer’s Disease, the neuronal axonal microtubule-binding TAU protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of TAU is accompanied by an abnormally high phosphorylation state of the TAU protein, and several downstream pathological consequences (e.g. loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of TAU sorting, missorting and associated pathologies have been addressed in rodent models, few studies have addressed human TAU in physiological disease-relevant human neurons. Suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, i.e., synapse formation, TAU isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we explain a simple way for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.

Axonal Tau sorting depends on the PRR2 domain and 0N4R-specific interactions hint at distinct roles of Tau isoforms in synaptic plasticity

“…Two different human iPSC lines were used in this study: 1) The WTC11 cell line carrying a doxycyclineinducible murine Ngn2 transgene ('WT iPSC-neurons' (Miyaoka et al, 2014;Wang et al, 2017;Zhang et al, 2013), and 2) a subclone of the above-mentioned line with CRISPR/Cas9-generated biallelic MAPT knockout ('MAPT-KO iPSC-neurons') (Buchholz et al, 2022). Both iPSC lines were cultivated as previously described (Bell et al, 2021;Buchholz, Bell-Simons, Cakmak, et al, 2024). Briefly, cells were cultured on GelTrex™-coated plates (Thermo Fisher) in iPS-Brew XF (StemMACS™, Miltenyi) supplemented with Anti/Anti™ solution (1X, Thermo Fisher).…”

“…WT iPSC-neurons were transduced with lentiviruses containing empty pUltra or pUltra-chili vectors for constitutive GFP or dTomato expression at different ages as previously described (Buchholz, Bell-Simons, Cakmak, et al, 2024). The virus particles were thawed on ice and mixed with pre-warmed and pH-adjusted maturation medium (50 % fresh, 50 % conditioned and sterile filtered).…”

“…Differentiation of both iPSC lines into cortical glutamatergic neuronal cultures was performed as previously described (Bachmann, Linde, et al, 2021;Buchholz, Bell-Simons, Cakmak, et al, 2024) with slight adaptations (Fig. S1a).…”

“…Tau HA construct expression in MAPT-KO iPSC-neurons 6-weeks-old MAPT-KO iPSC-neurons were transduced with lentiviruses containing pUltra-dox vectors with Tau HA constructs as previously described (Buchholz, Bell-Simons, Cakmak, et al, 2024) In brief, titrated virus particles were thawed on ice and mixed with pre-warmed and pH-adjusted maturation medium (50 % fresh, 50 % conditioned and sterile filtered). After cultures were washed once with DMEM/F12 medium, the virus mix was added for 16-24 h and replaced with doxycycline-containing (0.5 µg/ml) maturation medium (50/50).…”

“…In order to obtain a suitable neuronal model for our Tau sorting study, we differentiated human WTC11 iPSCs with a doxycycline-inducible Ngn2 cassette (Miyaoka et al, 2014;Zhang et al, 2013) with and without biallelic MAPT knockout (Buchholz et al, 2022) (Fig. S1a) into cortical glutamatergic neurons using protocols adapted from previous publications (Buchholz, Bell-Simons, Cakmak, et al, 2024;Tracy et al, 2022;Wang et al, 2017)).…”