nCounter immune profiling of RC17 hESC-derived neural progenitor cells (NPCs) and foetal ventral mesencephalon (fVM) dopamine cells

Description

Here we performed targeted transcriptional analysis of immune relevant genes in RC17-human embyronic stem cells (hESCs), RC17-hESC derived neural progenitor cells (NPCs) and mature dopamine neurons (DAn), differentiated according to Nolbrant et al. 2017, with comparison to human primary foetal ventral mesencephalon. All cell types were analysed either without treatment (NT) or after 24hr exposure to 5 ng/ml interferon gamma (IFNg) stimulation. The RC17-hESC derived NPCs are currently being investigated in the STEM-PD clinical trial as a dopamine cell replacement for Parkinson's disease (NCT05635409), here we utilised the Nanostring nCounter Human Immunology V2 panel, plus an additional 20 gene codeset designed to capture additional genes related to T cell activation/inhibition and graft rejection, to measure changes in immune gene expression throughout the differentiation process from hESC to mature DAn and with comparison to previously engrafted, and well-tolerated, fVM tissue.

Steps to reproduce

500,000 hESCs (no differentiation), NPCs, DAn (differentiated as per Nolbrant et al 2017 for 16 or 45 days, respectively) or fVM were seeded into 12 well plates with the appropriate coating substrate (hESC 0.5 ug/cm2 laminin-521, NPCs 1 ug/cm2 laminin-111, DAn 0.01% geltrex in DMEM, fVM PDL/Laminin-2020) and medium. Cells were either left untreated or treated with 5 ng/ml IFNg for 24 hours. After 24 hours, supernatants were removed, cells washed once with PBS then collected using accutase (hESC, NPCs) or ETDA (DAn, fVM) and cell pellets were resuspended in 350 ul RLT buffer. RNA was then extracted as per manufacturer's protocol using the Qiagen RNeasy plus kit. RNA concentration and quality was measured using the Agilent Bioanalyser (Nano kit), then 100 ng RNA used for Nanostring nCounter. nCounter was performed using the Human Immunology V2 panel with a custom add-on, hybridisation performed for 20 hours and 555 FOV imaged. RCC files were plugged into the nSolver software and data exported as normalised counts.

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