Biomedical Engineering Reference
In-Depth Information
Recent advancements in regenerative medicine have enabled cardiomyocytes to be
regenerated from various stem cells such as embryonic stem cells and bone marrow
mesenchymal stem cells [1, 3]. We previously reported that cardiomyocytes can be
regenerated from bone marrow mesenchymal stem cells by exposing them to
5-azacytidine [23]. We have also reported that cardiomyocytes can be efficiently ob-
tained from murine embryonic stem cells by stimulating them with Noggin, an inhibi-
tor of bone morphogenetic proteins [38]. These regenerated cardiomyocytes have
automaticity and show spontaneous beating. Others have reported that transplantation
of primary cultured fetal or neonatal cardiomyocytes into an infarcted area improves
cardiac function [39]. Furthermore, the regenerated cardiomyocytes can be purified
from a mixture of stem cell-derived cells [16, 25]. It has been suggested that patients
with bradyarrhythmias could be treated by transplantation of regenerated cardiomyo-
cytes [20, 36]. The use of autologous bone marrow mesenchymal stem cells shows
particular promise for clinical application, because the absence of immunorejection
means that immunodepressant drugs are not required.
Action potentials in cardiomyocytes are regulated by a variety of ion channels.
The expression of these ion channels and the shape of the action potentials varies
with the location of the cardiomyocytes and their developmental stage [4, 24]. Be-
fore regenerated cardiomyocytes can be used for treatment of bradyarrhythmias, it
needs to be determined what types of ion channels they express, whether they ex-
press adrenergic receptors [6, 9], and whether they express connexin43 and can make
gap junctions to the adjacent host cardiomyocytes. The present study focused on
these issues and investigated whether bone marrow mesenchymal stem cell-derived
cardiomyocytes could be used as bio-pacemakers. We describe here the current
status of the application of bone marrow mesenchymal stem cell-derived cardiomyo-
cytes as bio-pacemakers, and discuss the remaining issues that need to be resolved.
2 Materials and Method
2.1 Cell Culture and Cardiomyocyte Induction
The bone marrow mesenchymal stem cells used were cardiomyogenic (CMG) cells,
prepared as described previously [23]. Cells were cultured on 60 mm dishes in Iscove's
modified Dulbecco's medium (IMDM; GIB-CO/Invitrogen, Carlsbad, CA, USA). To
induce differentiation, cells were treated with 3 µmol/l of 5-azacytidine for 24 h and
then maintained for several weeks. After 5-azacytidine, cardiomyocytes comprised
approximately 20% of the cells, as described previously. The CMG cardiomyocytes
began to spontaneously contract 2-3 weeks after 5-azacytidine treatment.
2.2 Action Potential Recording
Electrophysiological studies were performed in IMDM containing (in mmol/l) 1.49
CaCl
2
, 4.23 KCl, and 25 HEPES (pH 7.4). Cultured cells were placed on the stage of
an inverted phase contrast microscope (Diaphoto-300; Nikon) at 23°C. Action poten-
tials were recorded using conventional glass microelectrodes filled with 3M KCl (DC
