Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells.
- Author:
Hye Won CHO
1
;
Kyoung Rae KO
;
Mi Kyoung KIM
;
Jae Ik LEE
;
Su Il SIN
;
Dong Hyung LEE
;
Ki Hyung KIM
;
Kyu Sup LEE
Author Information
1. Department of Obstetrics and Gynecology, College of Medicine, Pusan National University, Korea. kuslee@pusan.ac.kr
- Publication Type:Original Article
- Keywords:
Inner cell mass (ICM);
Embryonic stem cell;
Fibroblast;
Feeder cell
- MeSH:
Alkaline Phosphatase;
Animals;
Blastocyst;
Embryoid Bodies;
Embryonic Stem Cells*;
Embryonic Structures;
Family Characteristics;
Feeder Cells*;
Fibroblasts*;
Humans*;
Karyotype;
Mice*;
RNA, Messenger;
Skin;
Trypsin
- From:Korean Journal of Fertility and Sterility
2005;32(2):133-147
- CountryRepublic of Korea
- Language:Korean
-
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.
