SmartFlare^TM is a reliable method for assessing mRNA expression in single neural stem cells

Abstract

BACKGROUND One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlare TM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders. AIM To investigate the potential of SmartFlare TM in determining time-dependent mRNA expression of prominin 1 ( CD133 ) and octamer-binding transcription factor 4 ( OCT4 ) in single living cells through differentiation. METHODS Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlare TM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlare TM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells. RESULTS In agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes. CONCLUSION SmartFlare TM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Aquaporin‐4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment

“…Differently from the method described by Duval (Duval et al, 2002) and Scemes (Scemes et al, 2003), neural stem cell subculture was optimized by dissociating neurospheres with Accutase Cell Dissociation Reagent (Thermo, Waltham, MA, USA) for 15 min at room temperature (RT), followed by addition of the culture medium to halt enzymatic activity. The use of Accutase reagent enhances the dissociation of neurospheres into single cells and promotes neurosphere reformation, as evidenced by previous studies (Diana et al, 2021;Gonmanee et al, 2021;Panchision et al, 2007). Each neurosphere preparation was cultured for a maximum of 1 month, equivalent to four cell passages.…”