Highly efficient generation of glutamatergic/cholinergic NT2-derived postmitotic human neurons by short-term treatment with the nucleoside analogue cytosine β- d -arabinofuranoside

Abstract

The human NTERA2/D1 (NT2) cells generate postmitotic neurons (NT2N cells) upon retinoic acid (RA) treatment and are functionally integrated in the host tissue following grafting into the rodent and human brain, thus representing a promising source for neuronal replacement therapy. Yet the major limitations of this model are the lengthy differentiation procedure and its low efficiency, although recent studies suggest that the differentiation process can be shortened to less than 1 week using nucleoside analogues. To explore whether short-term exposure of NT2 cells to the nucleoside analogue cytosine β-d-arabinofuranoside (AraC) could be a suitable method to efficiently generate mature neurons, we conducted a neurochemical and morphometric characterization of AraC-differentiated NT2N (AraC/NT2N) neurons and improved the differentiation efficiency by modifying the cell culture schedule. Moreover, we analyzed the neurotransmitter phenotypes of AraC/NT2N neurons. Cultures obtained by treatment with AraC were highly enriched in postmitotic neurons and essentially composed of dual glutamatergic/cholinergic neurons, which contrasts with the preferential GABAergic phenotype that we found after RA differentiation. Taken together, our results further reinforce the notion NT2 cells are a versatile source of neuronal phenotypes and provide a new encouraging platform for studying mechanisms of neuronal differentiation and for exploring neuronal replacement strategies.

Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy

“…No. MAB2166, Millipore), or Choline acetyltransferase (ChAT) polyclonal antibody 46 (1:2000, Cat. No.…”

Data for the morphometric characterization of NT2-derived postmitotic neurons

“…NT2 progenitors were cultured and induced to differentiate into NT2N postmitotic neurons using either cytosine β-D-Arabinofuranoside (AraC) or retinoic acid (RA) as previously described in our laboratory [1] , thus obtaining cultures highly enriched in AraC/NT2N or RA/NT2N postmitotic neurons. Because β-III tubulin was expressed in cell bodies and throughout all neurites of differentiated AraC/NT2N and RA/NT2N cells, delivering at the same time high contrast [1] , we chose an antibody against β-III tubulin as the primary marker for measurements of area of neuronal body and neurite length in both neuronal phenotypes. Additionally, AraC-treated cultures were co-immunolabeled with an antibody against the neuronal marker NeuN/Fox-3, which served to establish a double criterion classify cells as neuronal or non-neuronal (see below).…”

“…The data from this analysis are shown in González-Burguera et al (2016, Fig. 3) [1] and served to assess whether phenotypes classified as neuronal and non-neuronal after AraC-induced differentiation were morphometrically distinguishable.…”

“…Pluripotent NTERA2/D1 (NT2) human teratocarcinoma cells induced to differentiate into postmitotic neurons (NT2N neurons) by either short-term treatment (6 days) with cytosine β-D-arabinofuranoside (AraC) [1] , [2] ( Fig. 1 A–F) or long-term treatment (4 weeks) with retinoic acid (RA) [1] , [3] , [4] ( Fig. 1 G–J) were morphometrically analyzed.…”

“…Our data provide a methodological and conceptual framework for future investigations aiming at distinguishing neuronal phenotypes on the basis of morphometric analysis. Data presented here are related to research concurrently published in “Highly Efficient Generation of Glutamatergic/Cholinergic NT2-Derived Postmitotic Human Neurons by Short-Term treatment with the Nucleoside Analogue Cytosine β-D-Arabinofuranoside” [1] .…”