Human neurospheres: From stained sections to three-dimensional assembly

Abstract

Human neurospheres are free-floating spherical clusters generated from a single neural stem cell and comprising cells at different stages of maturation in the neuronal and glial lineages. Although recent findings have disproved the original idea of clonally derived neurospheres according to the paradigm of one stem cell -one neurosphere, they still represent a valid model for growing neural stem cell cultures in vitro. While the immunocytochemical approach to the identification of stem cells, progenitor cells, and mature cells has been extensively used, scant data are available about the ultrastructural arrangement of different cell types within the neurosphere. This paper provides, by means of scanning electron microscopy, some new insights into the three-dimensional assembly of human neurospheres, trying to correlate some parameters such as cell density, shape and growing strategies with the immunolocalization of some antigens such as nestin, GFAP, α-internexin and βIII-tubulin. The major findings from this study are: a) regardless of the stage of in vitro maturation, the growth of the spheres is the result of mitotic divisions producing the aspect of an irregular budding mechanism in the outermost layer look like; b) analysis of the volumetric composition of the inner core has revealed the presence of two alternative shape pattern (pyramidal vs rounded cells) possibly related to both the ongoing maturation stages and GFAP and internexin expression.

Solving the Issue of Ionizing Radiation Induced Neurotoxicity by Using Novel Cell Models and State of the Art Accelerator Facilities

“…As a more realistic model system, neurospheres (NS), i.e., three-dimensional (3D) aggregates of several neural and neuronal progenitor types, derived from NSCs in suspension, were established. Within the NS, neural cells are able to selfrenew, generate various neuronal and glial subtypes at different stages of maturation [39] and display neuronal function in the form of spontaneously generated action potentials [40]. An organ-like microenvironment with some degree of structural or organizational integrity can be achieved, as shown by Merz et al [41], by culturing rodent and human tissue slices of about 300 µm at an air-liquid interface.…”

MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies

“…The final result was the prevention of spatial and learning deficits typical of AD [ 45 ]. For in vitro experiments [ 46 ], the extracellular vesicles isolated from hiPSCs and enriched with miR-133, -155, -221 and -34a conferred neuroprotection to Aβ42 oligomer-treated cortical spheroids [ 47 , 48 ]. With regard to remyelination strategy, which could positively impact MS, rat environmental enriched exosomes have been found to be upregulated in miR-219 content, fostering the increase in myelin but reducing the observed oxidative stress because of the dramatic consequences on the number of oligodendrocyte precursors and stem cells [ 49 ].…”

“…Along with regulating several biophysiological processes within NSCs, miRNAs are critically responsible for the control of cell fate in neural CSCs harbored in tumors of both neural and neural crest origin. These CSCs, considering the core cell subset of these tumors, express the NSC lineage markers nestin, prominin-1 (CD133), nanog homeobox (NANOG), sex determining region Y-box 2 (SOX2), hematopoietic cell E/L-selectin ligand (HCELL/CD44), octamer-binding transcription factor 4 (OCT4), bear a marked ability to form neurospheres in particular conditions [ 47 , 48 ], and are characterized by a tumor-initiating and self-renewal capacity through asymmetric cell division [ 3 , 66 , 67 , 68 ]. Moreover, the hallmarks of CSCs include high proliferation rate, multi-lineage differentiation potential, as well as great tendency for cell migration, invasiveness, and metastasis [ 69 , 70 , 71 ].…”

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells

“…To answer to some developmental issues related to the expression of the transcription factor Octamer-Binding Transcription Factor 4 ( OCT4 ), involved in the differentiation process of human neurospheres in a time-dependent fashion[ 15 , 16 ], the mRNA pattern of OCT4 at single-cell level was analyzed from 3 to 30 d in vitro (DIV) using specific SmartFlare TM probes to assess a possible downregulation strictly linked to cellular maturation from stem/progenitor to neural phenotype. In parallel, a SmartFlare TM probe for Prominin 1 ( CD133) , encoding for a transmembrane glycoprotein widely recognized as a marker of neural progenitor cells, was tested[ 17 , 18 ].…”