Development of human embryonic stem cell derivation.
OBJECTIVE
To establish human embryonic stem (hES) cells from human embryos.


METHODS
Experimental study.


METHODS
Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University.


METHODS
Abnormal and normal fertilization embryos were cultured in vitro until reaching blastocyst stage. Four different methods for isolation of ICMs were used. Immunosurgery, mechanical isolation, laser assists, and whole blastocyst culture were performed. The feeder layers used in the present study were fibroblasts, isolated from either mouse or human. Mechanical splitting of ICM outgrowths or hES-like cells was performed for propagation of cells. Characterization of hES-like cells was conducted by morphology, detection of immunostaining of Oct-4, and enzymatic activity of alkaline phosphatase (AP). HES-like cells were spontaneously differentiated through suspension culture of embryoid body (EB). Subsequent differentiation was done on gelatin-coated dishes.


METHODS
Establishment of hES cells.


RESULTS
By using abnormal fertilization embryos, 80.0% (8/10) of blastocysts were able to attach on the feeder layers, 50% (4/8) formed ICM outgrowths, but no hES-like cells were established. By using normal fertilization embryos, 84.6% (22/26) of blastocysts were able to attach on feeder layers, 18.2% (4/22) formed ICM outgrowths. One hES-like cell line was successfully established by using mechanical isolation of ICMs and human adult skin fibroblasts as feeder layers. This hES-like cells exhibited typical morphology of hES cells, positive staining for Oct-4 and AP. hES-like cells were able to form EB and differentiated into neural-like cells.


CONCLUSIONS
This is the first report in Thailand that hES-like cells can be isolated from normal development human embryos at blastocysts-stage using mechanical isolation of ICM and culture with human adult skin fibroblast as feeder layers.
Pruksananonda
Kamthorn
K
Reproductive Medicine Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. fmedkpn@md.chula.ac.th
Rungsiwiwut
Ruttachuk
R
Numchaisrika
Pranee
P
Ahnonkitpanich
Vichuda
V
Virutamasen
Pramuan
P
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000378:metabolism; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D015925:Cryopreservation; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D012867:Skin / Q000166:cytology; D013785:Thailand
D000469:Alkaline Phosphatase