Healthcare and Medicine Reference
In-Depth Information
Cultured
blastocyst
Isolated inner
cell mass
First plating
Cultured inner cell mass
9-15 days
Cultured
inner cell mass
dissociated . . .
Irradiated mouse
fibroblast feeder cells
Inner cell mass
Human
embryonic
stem cells
. . . and replated
onto new
feeder cells
7-10 days
Established
cultures
Passaged
Second plating to
establish colonies
(a)
HESC colony
Feeder cells
Feeder cells
HESCs
(b)
(c)
FIGURE 1.2
Derivation of human embryonic stem cells (hESCs). (a) The most common source of hESCs is
the ICM of the blastocyst. To initiate new hESC lines, the cells of the ICM are plated on a feeder
layer. As the ICM cells attach, they form colonies that can be isolated and passaged to derive
new hESC lines. (b) An example of an undifferentiated hESC colony. (c) An example of enzy-
matically passaged hESCs grown on irradiated mouse fibroblast feeder cells.
Typically, undifferentiated hESCs are maintained in culture as colonies
that are generally passaged once a week either mechanically or enzymati-
cally as small clusters of cells (FigureĀ 1.2B). 29,30 This method is preferred for
long-term maintenance of hESCs as it reduces the level and frequency of
stress associated with passaging and appears to minimize the occurrence of
karyotypic abnormalities. 31 Alternatively, hESCs can also be expanded enzy-
matically as a single-cell suspension for short periods without the appearance
of cells with an abnormal karyotype or changes detected by morphological
and low cytometric analysis (FigureĀ 1.2C). 29,32 It is possible that the incidence
of karyotypic changes occurs at the same frequency with both propagation
methods, although the absolute number of cell divisions that occur during
enzymatic passage may be higher than during mechanical passage, result-
ing in the genetically abnormal cells being more readily detected. 33 In both
culture systems, the hESCs maintain their compact morphology with a high
nuclear-to-cytoplasmic ratio and prominent nucleoli.
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