© 1996 American Heart Association, Inc.
Articles |
Vital Staining of Cardiac Myocytes During Embryonic Stem Cell Cardiogenesis In Vitro
From the Department of Physiology, School of Medicine, University of Michigan, Ann Arbor.
Correspondence to Dr Joseph M. Metzger, Department of Physiology, University of Michigan, School of Medicine, 7730 Medical Science II, Ann Arbor, MI 48109-0622.
Abstract Mouse embryonic stem (ES) cells differentiate in
vitro into a variety of cell types, including spontaneously contracting
cardiac myocytes. The primary aim of this work was to use vital stain
techniques for real-time detection of developing cardiac myocytes
in ES cell differentiation cultures. The -440 to +6 human cardiac
-actin promoter was used to direct expression of the
Escherichia coli reporter gene lacZ (pHCActlacZ)
into ES cell–derived cardiac myocytes during cardiogenesis in
vitro. Undifferentiated ES cells were electroporated with HCActlacZ
together with a plasmid containing the neomycin gene under the
direction of the phosphoglycerate kinase promoter, and stable
transformants were selected in G418. Individual clones were screened
for activation of lacZ gene expression in cardiac myocytes
developing in vitro. Results showed that expression of the HCActlacZ
reporter construct was activated very early during the ES cell
differentiation program, at a time point before the appearance of
spontaneous contractile activity. The earliest detection was at day 6
of differentiation, when 
25% of the differentiation cultures
expressed the reporter construct, with expression increasing to
70% at day 9 and continuing throughout the duration of spontaneous
contractile activity exhibited by the ES cell–derived cardiac
myocytes. Indirect immunofluorescence assays
provide evidence that expression was restricted to the cardiac myocytes
in culture. In the present study, we show vital staining of
transgene expression in living cardiac myocytes using lipophilic
fluorogenic ß-galactopyranoside substrates for real-time
detection of the reporter gene during continuous contraction of the ES
cell myocytes in vitro. The vital stain approach used in the
present study will permit the identification of differentiating ES
cells that are committed to the cardiac lineage for analysis of
gene expression at early time points of ES cell cardiogenesis and, in
addition, will aid in selecting genetically modified ES cell cardiac
myocytes for use in functional studies.
Key Words: contractility • myofilaments • gene expression • development
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