Educational Forum with Clinical Studies Current Science and Research

Katia Mareschi , Deborah Rustichelli, Valentina Comunanza, Roberta De Fazio, Cristina Cravero, Giulia Morterra,Barbara Martinoglio, Enzo Medico, Emilio Carbone, Chiara Benedetto and Franca Fagioli

Background aims
Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro.
Methods:
AF was harvested during routine pre-natal amniocentesis at 14–16 weeks of pregnancy. AF sample pellets were plated in α-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion.
Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction.
Results:
Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na+ channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na+ channels.
Conclusions:
These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.

Xanya Sofra Weiss

Xanya Sofra Weiss

T. Cho, J. H. Bae, H. B. Choi, S. Kim, J. G. McLarnon, H. Suh-Kim, S. U. Kim, CA and C. K. Min

We have characterized the pro¢le ofmembrane currents in an immortalized human neural stem cell line, HB1.F3 cells, using wholecell patch clamp technique.Human neural stemcell line generated from primary cell cultures of embryonic human telencephalon using a replication-incompetent retroviral vector containing vmyc expresses nestin, a cell type-speci¢c marker for neural stem cells.The human neural stemcells expressed both outward and inward Kþ currents with no evidence for Naþ currents.The density of the outward, delayed rectifying type Kþ current was 1.870.015 nA/pF, and that of the inwardly rectifying Kþ current was 0.3770.012 nA/pF (at 30mMof [Kþ]o). In order to induce neuronal di¡erentiation of the neural stem cells, a full-length coding region of NeuroD, a neurogenic transcription factor, was transfected into HB1.F3 cells. Introduction of NeuroDi nduced expression of Naþ currents with the current density of 0.04270.011nA/ pF. The presence of two types of Kþ currents and expression of Naþ currents induced byNeuroDap pear to re£ect the characteristic physiological features of human neural stemcells.

Xanya Sofra Weiss

Xanya Sofra Weiss

Researchers from Johns Hopkins have discovered the presence of functional ion channels in human embryonic stem cells (ESCs). These ion channels act like electrical wires and permit ESCs, versatile cells that possess the unique ability to become all cell types of the body, to conduct and pass along electric currents. If researchers could selectively block some of these channels in implanted cells, derived from stem cells, they may be able to prevent potential tumor development. The paper appears Aug. 5 online in the journal Stem Cells. “A major concern for human ESC-based therapies is the potential for engineered grafts to go haywire after transplantation and form tumors, for instance, due to contamination by only a few undifferentiated human ESCs,” says Ronald A. Li, Ph.D., an assistant professor of medicine at The Johns Hopkins University School of Medicine and senior author of the study. “Our discovery of functional ion channels, which are valves in a cell’s outer membrane allowing the passage of charged atoms, the basis of electricity, provides an important link to the differentiation, or maturation, and cell proliferation, or growth of human ESCs.” Because human ESCs can potentially provide an unlimited supply of even highly specialized cells, such as brain and heart cells, for transplantation and cell-based therapies, they may provide an ultimate solution to limited donor availability.

Xanya Sofra Weiss

Xanya Sofra Weiss

Maddalena Illario, Anna Lina Cavallo, Sara Monaco, Ennio Di Vito, Frank Mueller, Luigi A. Marzano, Giancarlo Troncone, Gianfranco Fenzi, Guido Rossi, and Mario Vitale

Werecently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium (Ca2 )/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FNinduced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2 concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2 concentration were
mediated by FN binding to v 3 integrin. Inhibition of the Ca2 /CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/ Mek/Erk and Ca2 /CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2 /CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2 /CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered. (J Clin Endocrinol Metab 90: 2865–2873, 2005)

Xanya Sofra Weiss

Xanya Sofra Weiss