Mathematical model for NF-kappaB-driven proliferation of adult neural stem cells

Neural stem cells (NSCs) are early precursors of neuronal and glial cells. NSCs are capable of generating identical progeny through virtually unlimited numbers of cell divisions (cell proliferation), producing daughter cells committed to differentiation. Nuclear factor kappa B (NF-kappaB) is an inducible, ubiquitous transcription factor also expressed in neurones, glia and neural stem cells. Recently, several pieces of evidence have been provided for a central role of NF-kappaB in NSC proliferation control. Here, we propose a novel mathematical model for NF-kappaB-driven proliferation of NSCs. We have been able to reconstruct the molecular pathway of activation and inactivation of NF-kappaB and its influence on cell proliferation by a system of nonlinear ordinary differential equations. Then we use a combination of analytical and numerical techniques to study the model dynamics. The results obtained are illustrated by computer simulations and are, in general, in accordance with biological findings reported by several independent laboratories. The model is able to both explain and predict experimental data. Understanding of proliferation mechanisms in NSCs may provide a novel outlook in both potential use in therapeutic approaches, and basic research as well.

Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Adult palatum as a novel source of neural crest-related stem cells

Somatic neural and neural crest stem cells are promising sources for cellular therapy of several neurodegenerative diseases. However, because of practical considerations such as inadequate accessibility of the source material, the application of neural crest stem cells is strictly limited. The secondary palate is a highly regenerative and heavily innervated tissue, which develops embryonically under direct contribution of neural crest cells. Here, we describe for the first time the presence of nestin-positive neural crest-related stem cells within Meissner corpuscles and Merkel cell-neurite complexes located in the hard palate of adult Wistar rats. After isolation, palatal neural crest-related stem cells (pNC-SCs) were cultivated in the presence of epidermal growth factor and fibroblast growth factor under serum-free conditions, resulting in large amounts of neurospheres. We used immunocytochemical techniques and reverse transcriptase-polymerase chain reaction to assess the expression profile of pNC-SCs. In addition to the expression of neural crest stem cell markers such as Nestin, Sox2, and p75, we detected the expression of Klf4, Oct4, and c-Myc. pNC-SCs differentiated efficiently into neuronal and glial cells. Finally, we investigated the potential expression of stemness markers within the human palate. We identified expression of stem cell markers nestin and CD133 and the transcription factors needed for reprogramming of somatic cells into pluripotent cells: Sox2, Oct4, Klf4, and c-Myc. These data show that cells isolated from palatal rugae form neurospheres, are highly plastic, and express neural crest stem cell markers. In addition, pNC-SCs may have the ability to differentiate into functional neurons and glial cells, serving as a starting point for therapeutic studies.

Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.