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

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
normoxic atmosphere (20% O 2 ) and 5% CO 2 at 37ºC. Passage was
done every 3 or 4 days.
Umbilical cord and placenta-derived mesenchymal cells were isolated by
explant culture as described previously 2 . Umbilical cord and chorionic
plate were chopped into small pieces, 5-8 mm 3 blocks, and put on 10 cm
dishes with 10 ml hi-glucose Dulbecco Modified Eagle Medium (DMEM-
H) with 10 % fetal bovine serum, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 1µg/mL of amphotericin B (Gibco). Mesenchymal cells
came out within one or two weeks, and passage was done once a week.
2. Calculation of cardiomyogenic transdifferentiation efficiency in vitro
It is difficult to measure accurately the population of spontaneously
contracted EGFP-positive hAMCs, since they usually gather together and
generate a colony of cells with thick cell layers in the co-culture system.
Contraction of each cell in the colonized EGFP-positive cells was usually
unclear because it was difficult to detect the margin of each cell and to
determine which cells were beating and which cells were not. To enable
this, isolation of the cell is necessary. Immediately after enzymatic
isolation of the co-cultured hAMCs, we could observe spontaneouslybeating
EGFP-positive hAMCs. However, the number of beating cells
was significantly reduced due to the enzymatic isolation. Therefore, we
defined cardiac troponin-I positive cells as the cells transdifferentiated
into cardiomyocytes. In order to stain the intracellular protein structure,
we tried using triton-X to permeate the cellular membrane. This appeared
to reduce the specific gravity of the cell; therefore, we could not rinse the
antibody from the floating isolated hAMCs by centrifugation, etc. Thus,
we could not use the FACS system in this protocol, but devised an
original method to evaluate the cardiomyogenic induction rate.
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