J Med Assoc Thai 2009; 92 (4):443

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Development of Human Embryonic Stem Cell Derivation
Pruksananonda K Mail, Rungsiwiwut R , Numchaisrika P , Ahnonkitpanich V , Virutamasen P

Objective: To establish human embryonic stem (hES) cells from human embryos.

Design: Experimental study.

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

Material and Method: 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). HESlike
cells were spontaneously differentiated through suspension culture of embryoid body (EB). Subsequent
differentiation was done on gelatin-coated dishes.

Main outcome measure: 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

Conclusion: 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.

Stem cell, Cultured, Embryo, Fibroblasts, Human, IVF

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