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Fig. 1. In vitro heart induction system using embryonic pluripotent cells. (A) The
dissociation/reaggregation protocol for in vitro heart induction. The cell adhesion of mid-blastula
animal caps were loosened in Ca2+-free medium. Dissociated (by gentle pipetting) cells began
to form a spherical reaggregate in the medium containing Ca2+ and an appropriate concentration
of activin. (B) Xenopus cardiac troponin I (XTnIc), a marker gene for myocardial differentiation,
expressed specifically in myocardium of a normal 3-day-old embryo (upper). The dissociated
animal cap cells formed spherical reaggregates, regardless of whether they had been treated
with activin. However, untreated reaggregates showed negative staining of the XTnIc signal
after they were cultured for 3 days (lower). XTnIc signals were detected only in activin-treated
reaggregates (middle). Note the XTnIc signals (arrows) at the tubular hearts on the transverse
sections of the normal embryo and the activin-treated reaggregate. Scale bar, 0.5 mm. (C)
Activin frequently induced beating hearts at concentrations higher than 100 ng/ml. The
frequency of beating-heart formation reached 100% when the animal cap cells were treated
with 1,000 ng/ml activin for 3 h. Each reaggregate was comprised of 10 animal caps (approx.
2,000 cells). (D) The number of cells contained in a reaggregate also affected heart formation.
The dissociated cells were treated with 100 ng/ml activin for 5 h in this experiment. The
frequency of heart formation reached 100% when 5 animal caps (approx. 1,000 cells) were
contained in a reaggregate. The number on the top of each column refers to the number of
reaggregates with beating hearts per the total number of reaggregates. |
