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

Tsuji et. al. amniotic membrane-derived stem cell
(2009/205260-R3)
Supplemental Material and Methods
1. The isolation of the amniotic membrane-derived mesenchymal cells
Amniotic membrane consists of fetal amniotic membrane and maternal
deciduas. Deciduas are a part of uterine endometrium, which also
contains a lot of cardiac precursor-like cells (Hida et al., Stem Cells,
2008; 26(7): 1695-1704). The speed of population doubling of maternal
endometrial cells was significantly greater than hAMCs (18.2 ± 1.7 PDs
at 40 days in endometrial cells vs 6.68 ± 1.67 PDs at 40 days in hAMCs).
Therefore, in our preliminary data, even if there was a small amount of
contamination of maternal cells at the start of the culture, they quickly
overcame the hAMCs and, finally, the majority of the cells became the
maternal endometrial cells. Therefore, a human amniotic membrane was
collected after delivery of a male neonate in order to check the maternal
cell contamination in culture by the karyotype. At the 2 nd passage and
10 th passage of hAMCs, the CEP X/Y DNA Prove Kit (Vysis) was used to
determine the proportion of XX and XY cells in accordance with the
manufacturer!s suggestions.
hAMCs were isolated according to the method previously described 1 with
slight modification. Avascular transparent amniotic membrane was
peeled off from maternal placenta and deciduas, rinsed with phosphatebuffered
saline (PBS), chopped into small pieces, and incubated in 2.4
U/mL dispase II (Roche Diagnostics, 4942078) at 37ºC for 55 minutes.
The membranes were then incubated in 0.75mg/mL type-II collagenase
(Worthington Biochemical, NJ, USA) for 60 minutes at 37ºC. Isolated
cells were rinsed and seeded on a 10 cm culture dish with Mesenchymal
Stem Cell Basal Medium (MSCBM, LONZA, PT-3238). After two or three
days, the cultured dishes became subconfluent in a humidified and
Suppl 5