Tsuji et. al. amniotic membrane-derived stem cell (2009/205260-R3 ...

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
of the EGFP-positive cardiomyocytes was calculated in every tissue slice.
In the present study, the size of EGFP-positive cardiomyocytes are the
same as the host cardiomyocytes; therefore, the average size of the
EGFP-positive cardiomyocytes was postulated as normal mammalian
ventricular cardiomyocytes (circular cylinder 100 µm in length and 11 µm
in radius, cell volume is calculated as 37.994 pL, 3 . Because the
cardiomyocyte array is along a random direction, the incidence of
appearance in a tissue section is postulated to be the same as the
incidence of appearance, in a single slice, of a sphere of the same
volume (radius 20.9 µm). This suggests that, if we sliced the tissue at
every 41.8 µm, we could observe every EGFP-positive cell in the heart.
Therefore, we assume the number of surviving EGFP-positive
cardiomyocytes to be equal to (total count of EGFP-positive
cardiomyocytes from every slice) x (360/41.8). The rate of surviving
EGFP-positive cardiomyocytes was defined as the number of the
surviving EGFP-positive cardiomyocytes divided by the number of
injected hAMCs.
5. Isolation of transdifferentiated cardiomyocytes and FISH experiment
Method for isolation of cardiomyocytes was shown in our previous
paper 4 , but with slight modification. Excised heart was perfused
retrogradely, with nominally Ca 2+ free Tyrode!s solution (described later)
with 0.1% bovine serum albumin (BSA, fraction-V, Gibco) for 3 minutes,
and then switched to an enzyme solution containing 0.8 g/L of type-II
collagenase (Worthington Biochemical, NJ, USA) and 0.2 % BSA for 25-
30 minutes. Next, the left ventricle was minced, incubated, and gently
agitated in enzyme solution containing 0.8 g/L of the collagenase, 3 %
BSA, and 0.3 mmol/L CaCl 2 . Incubation with fresh enzyme solution was
repeated 7-9 times at 10-minute intervals. The supernatant from each
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