Petrova R.R., Mezhevikina L.M., Osipenko M. A., Fesenko E.E.
(Institute of Biophysics of Cell, Russian Academy of Sciences, 142290, Pushchino, Moscow region, Russia, e-mail: rushap@rambler.ru)
The increased attention to mammalian embryonic stem cells (ESC) is caused by reason, that these cultures are the source of differentiated cells used in fundamental and applied researches. Pluripotent embryonic stem cell lines are capable for spontaneous differentiations into other type of cells and tissues (Ventura et al., 2004; Wobus, Boheler 2005). Various techniques of cultivation on the feeder layers and chemical inductors are used for the certain phenotype of ESC receiving (Reubinoff et al., 2000; Itskovitz-Eldor et al., 2000; Boheler et al., 2002; Ventura et al., 2004). As a rule, these ESC differentiations in vitro are connected with formation of embryoid bodies (EB).
It worth mentioning, that induction of differentiation into EB is connected with formation of heterogeneous population with little part of certain phenotype cells (Boheler et al., 2002). At present time the heterogeneity of ESC is essential limitation for use in applied medicine. The aim of our work was receiving of cardiomyocyte type from ESC without the EB formation. For that purpose the ESC were prolonged cultivated with the recombinant LIF protein from Сos-1 cells transfected by pET28b plasmid (Invitrogene) with mouse lif gene (Petrova et al., 2005). The commercial mouse LIF recombinant protein (ICN) was used as a control.
Fig. 1. The morphology of ESC R1 colonies grown in the medium with the recombinant cytokine LIF-Cos: а) - colony overview through 3 and b) - 7 cultivation days, c) - differentiated colonies, d) - endogenous alkaline phosphatase activity on 24 cultivation day. Arrows point at foci.
In contrast to LIF-ICN, the recombinant Lif-Cos has a distinctive feature: it increases the ability of mouse ESC to survive in form of colonies without formation of embryoid bodies (fig. 1, a, b). At prolonged cultivation with Lif-Cos, the autonomous contractive activity foci of about 1.55±0.4 millimeter2 (1.6х104±0.29 cells, n = 6) size, are appearing in plurypotent colonies on 20-26 cultivation days. At cytochemical determination of endogenous alkaline phosphatase (AP) activity, nonuniform coloration of such colonies was shown (fig. 1, d). The AP activity in contractive region was reduced, that points on process of cell differentiation.
According to our observations, cells of interface between more compact central mass of pluripotent cells with high AP activity and peripheral monolayer are those that differentiate into contractive activity ones (fig. 1, d). Furthermore, several contractive foci were registered in the same colonies with different sizes and frequency and amplitude of contractions (fig. 1, c).
For identification of type of differentiated cells we used the cardioactive substance - isoproterinol (IP) (10-7-10-5 М). In mammalian embryonic cardiomyocites isoproterinol selectively acts on ?-adrenergic receptors and affects contractive rhythm by activation of ion channel, membrane transport systems and myofilament proteins (Boheler et al., 2002; He et al., 2003).
Fig. 2. The differentiation of ESC R1 on the 24th day of cultivation in medium with recombinant LIF-Cos: a - undifferentiated colony of pluripotent cells, b-c - cardiac ?-actin in differentiated cells. Lens magnification 20 x.
The dynamics of contractive activity of differentiated cells in control (1) and at 10-7 М isoproterinol influence (2).
The maximal response of differentiated mouse ESC on ?-adrenergic stimulation appeared 2-3 minutes after addition of 10-7 М isoproterinol to cultivation medium. At isoproterinol influence the contractive frequency is increased from 19±1.2 to 58±2.3 beats per minute (fig. 2, B). This reaction is typical for cardiac cells with ?-adrenoreceptors expression. Determination with by monoclonal antibodies on mouse cardiac ?-actin proved cardiomyocite differentiation cell type (fig. 2, A). More early predecessors of cardiac cells have small size and round shape (fig. 2, b). The specific cells with sarcomere organization and ability for contraction are forming in the course of their development in vitro (fig. 2, c).
The presence of ?-adrenoreceptors and myofilament cardiac ?-actin in mouse ESC is evidence of activation of cardiac differentiation processes occurring after prolonged cultivation with the recombinant LIF-Cos protein. According to results of LIF - regulation investigations, LIF protein effects on different transduction ways including those responsible for differentiation of pluripotent cells (Auernhammer et al., 2001; Boiani, Scholer, 2005; Izumi et al., 2006). The mouse ESC colonies with retractive activity are an experimental model for revealing of cell and molecular mechanisms of cardiac differentiation, for electrophysiological investigations, as well as cardioactive drugs screening.
The work was supported by grants «RFFI № 05-04-49521» and «SS-1842. 2003.4»
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