Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells / 대한불임학회지
Korean Journal of Fertility and Sterility
;
: 133-147, 2005.
Article
in Korean
| WPRIM
| ID: wpr-60747
ABSTRACT
OBJECTIVES:
This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells.METHODS:
When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker.RESULTS:
We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46, XX karyotype.CONCLUSIONS:
The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Skin
/
Blastocyst
/
RNA, Messenger
/
Trypsin
/
Family Characteristics
/
Embryonic Structures
/
Alkaline Phosphatase
/
Embryonic Stem Cells
/
Embryoid Bodies
/
Feeder Cells
Type of study:
Prognostic study
Limits:
Animals
/
Humans
Language:
Korean
Journal:
Korean Journal of Fertility and Sterility
Year:
2005
Type:
Article
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