Development of human embryonic stem cell derivation.

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). HES-like 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 cells. 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.

Effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocysts and subsequent derivation of embryonic stem (ES) cells.

OBJECTIVE: To determine the effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocyst and the efficiency of embryonic stem (ES) cell derivation. DESIGN: Experimental study SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University MATERIAL AND METHOD: In vivo fertilized zygotes were collected and subjected to in vitro culture in potassium simplex optimized medium (KSOM) containing 1,000 unit/ml LIF. The developmental ability of the zygote to blastocyst-stage and the cell numbers in blastocysts were evaluated Expanded blastocysts developed in different culture media were subsequently subjected to ES cell derivation. MAIN OUTCOME MEASURE (s): The influence of LIF on the quality of and the total cell numbers of blastocyst developed in vitro. RESULTS: Supplementation of LIF in KSOM increased the rate of hatching blastocysts (63.8% vs. 53.7%; p < 0.05) and total cell numbers (91.4 +/- 15.0 vs. 85.1 +/- 7.7; p < 0.05) compared to KSOM alone. ES cells were obtained 66.7% from blastocysts developed in KSOM-LIF versus 41.7% in KSOM (p > 0.05). Established ES cell lines showed typical colony and characteristics of pluripotent murine ES cells. CONCLUSION: LIF improved the quality of in vitro produced blastocysts but not enhanced ES cell derivation.

Conventional and novel methods for embryonic stem cell line derivation.

Cell therapy is the promising therapeutic tool for the next decade. "Regenerative Medicine" based on cell and tissue replacement therapy is proposed as a revolutionary approach to various chronic and incurable conditions. The first key step for successful cell therapy is the establishment of clinical grade human Embryonic Stem Cell (hESC) lines. This article provides a concise summary on conventional and novel methods for hESC line derivation. There is also discussion on progression, future direction and problems in hESC line development. In Thailand, more advance knowledge, skill, and technology are required to develop the first human embryonic stem cell line and step forward to make cell therapy a reality.