signature=62c105790f653bfef64dbbe6a3c283e4,Plasticity of Calcium Signaling Cascades in Human

ORIGINAL RESEARCH REPORTS

Plasticity of Calcium Signaling Cascades in Human Embryonic Stem Cell-Derived Neural Precursors

Oksana Forostyak,1Nataliya Romanyuk,2Alexei Verkhratsky,3Eva Sykova,2,4and Govindan Dayanithi 1,5

Human embryonic stem cell-derived neural precursors (hESC NPs)are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases.The Ca 2+ion is an important intracellular messenger essential for the regulation of various cellular functions.We investigated the role and physiology of Ca 2+signaling to characterize the functional properties of CCTL14hESC NPs during long-term maintenance in culture (in vitro).We analyzed changes in cytoplasmic Ca 2+concentration ([Ca 2+]i )evoked by high K +,adenosine-5￠-triphosphate (ATP),glutamate,g -aminobutyric acid (GABA),and caffeine in correlation with the expression of various neuronal markers in different passages (P6through P10)during the course of hESC differentiation.We found that only differentiated NPs from P7exhibited signi?cant and speci?c [Ca 2+]i responses to various stimuli.About 31%of neuronal-like P7NPs exhibited spontaneous [Ca 2+]i oscillations.Pharmacological and immunocytochemical assays revealed that P7NPs express L-and P/Q-type Ca 2+channels,P2X 2,P2X 3,P2X 7,and P2Y purinoreceptors,glutamate receptors,and ryanodine (RyR1and RyR3)receptors.The ATP-and glutamate-induced [Ca 2+]i responses were concentration-dependent.Higher glutamate concentrations (over 100m M)caused cell death.Responses to ATP were observed in the presence or in the absence of extra-cellular Ca 2+.These results emphasize the notion that with time in culture,these cells attain a transient period of operative Ca 2+signaling that is predictive of their ability to act as stem elements.

Introduction

H

uman embryonic stem cells (hESCs)are pluripotent cells derived from the inner cell mass of a preimplanta-tion embryo [1].In vitro,these cells are able to maintain a normal euploid kariotype,differentiate into derivatives of all 3germ layers,and proliferate extensively [2,3].These prop-erties make them unique candidates for cell transplantation,research into growth factors and early human development,and for drug discovery.Substantial progress has been made recently in the differentiation of hESCs into a neuronal phe-notype [3–5],this being a promising strategy for cell-based therapy of central nervous system injuries and neurodegen-erative diseases.However,to date,several important ques-tions remain unanswered:(1)when and at what stage of differentiation should cells be transplanted;(2)what are the functional properties (ion channels,receptors,and second messengers)of these cells and how are they regulated;and (3)how compatible are these properties with the physiological or pathological environment at the site of transplantation and

treatment?Hitherto,with a few exceptions,the quality of stem cells is generally evaluated by determining the expression of various genes and key proteins during the process of differ-entiation;these however,although being present in the cell,may be physiologically inactive.Therefore,the aim of this study was,for the ?rst time,to determine and characterize Ca 2+signals activated by physiological stimulation of neural precursors (NPs)derived from hESCs.

Ca 2+is a ubiquitous second messenger involved in the regulation of almost all known cellular processes and,above all,in de?ning the life and death of every cell [6–10].Signals mediated by Ca 2+are fundamental for fertilization,cell differentiation,proliferation [11],intercellular signaling,transcription factor activation,and various death programs including necrosis and apoptosis [12].Ca 2+can enter the cytoplasm from 2sources:either by an in?ux via plasma-lemmal voltage-operated and receptor-operated Ca 2+chan-nels (VOCC and ROCC respectively)or by release from intracellular stores,such as the endoplasmic reticu-lum,through endomembrane Ca 2+channels classi?ed as

Departments of 1Molecular Neurophysiology,and 2Neuroscience,Institute of Experimental Medicine,Academy of Sciences of the Czech Republic,Prague,Czech Republic.3

School of Biological Sciences,University of Manchester,Manchester,United Kingdom.4

Department of Neuroscience,Second Medical Faculty,Charles University,Prague,Czech Republic.5

Institut National de la Sante

′et de la Recherche Me ′dicale,Unite ′de recherche U710,Universite ′Montpellier 2,Montpellier;and Ecole Pratique des Hautes Etudes,Paris,France.

STEM CELLS AND DEVELOPMENT Volume 22,Number 10,2013óMary Ann Liebert,Inc.DOI:10.1089/scd.2012.0624

1506