Cell Renewing in Neuroblastoma: Electrophysiological and Immunocytochemical Characterization of Stem Cells and Derivatives

Abstract

We explored the stem cell compartment of the SH-SY5Y neuroblastoma (NB) clone and its development by a novel approach, integrating clonal and immunocytochemical investigations with patch-clamp measurements of ion currents simultaneously expressed on single cells. The currents selected were the triad I HERG , I KDR , I Na , normally expressed at varying mutual ratios during development of neural crest stem cells, from which NB derives upon neoplastic transformation. These ratios could be used as electrophysiological clusters of differentiation (ECDs), identifying otherwise indistinguishable stages in maturation. Subcloning procedures allowed the isolation of highly clonogenic substrate-adherent (S-type) cells that proved to be p75-and nestin-positive and were characterized by a nude electrophysiological profile (ECDS 0 ). These cells expressed negligible levels of the triad and manifested the capacity of generating the two following lineages: first, a terminally differentiating, smooth muscular lineage, positive for calponin and smooth muscle actin, whose electrophysiological profile is characterized by a progressive diminution of I HERG , the increase of I KDR and I Na , and the acquisition of I KIR (ECDS 2 ); second, a neuronal abortive pathway (NF-68 positive), characterized by a variable expression of I HERG and I KDR and a low expression of I Na (ECDN S ). This population manifested a vigorous amplification, monopolizing the stem cell compartment at the expense of the smooth muscular lineage to such an extent that neuronal-like (N-type) cells must be continuously removed if the latter are to develop.

Purging of the Neuroblastoma Stem Cell Compartment and Tumor Regression on Exposure to Hypoxia or Cytotoxic Treatment

“…The human neuroblastoma SY5Y clone and its derivatives were maintained in RPMI 1640 and cultured as previously reported (2).…”

“…We carried out a program of research aimed at revealing the process of cell renewing in neuroblastoma, a tumor of childhood characterized by an unpredictable clinical course and a remarkable plasticity, resulting in multiform phenotypes (1,2). A peculiar feature of this tumor is the tendency to regress from a malignant to a benign phenotype (ganglioneuroma or, rarely, a neurofibromatous mass; ref.…”

“…Our approach to this problem was based on the use of a human neuroblastoma adrenergic SH-SY5Y clone (henceforth referred to as SY5Y), taking advantage of our previous studies (2), which unraveled the renewal of this tumor by an integrated investigation, which coupled classic immunocytochemical procedures with sophisticated electrophysiologic measurements done with the patch-clamp technique. The latter permits quantitative measurements of the clusters of ion currents expressed on the surface of single cells, providing a powerful tool to identify cell types and differentiation stages.…”

“…In this approach, it was possible to use the grouped currents in the same way as cluster of differentiation (CD) antigens are widely used in cell biology and immunology (4,5). By analogy, we introduced the term electrophysiologic cluster of differentiation # (ECD#) to describe the quantitative expression of a group of ion channels on the cell under investigation, where # is a signal identifying a determinate stage of differentiation along a specific pathway (2).…”

Diversity of Ion Channels in Human Bone Marrow Mesenchymal Stem Cells from Amyotrophic Lateral Sclerosis Patients

“…Ion channels are widely expressed in proliferative cells which can modulate the proliferation of cells by affecting the cell cycle (MacFarlane & Sontheimer, 2000;Ouadid-Ahidouch et al, 2004;Biagiotti et al, 2006). Recent studies from our group and others have demonstrated that multifunctional ion channels are heterogeneously expressed in various species of MSCs (Li et al, 2005;Deng et al, 2006;Li et al, 2006;Park et al, 2007;Tao et al, 2007).…”