Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation.

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.

Correlation of colposcopic anogenital findings and overall assessment of child sexual abuse: prospective study.

OBJECTIVE: To examine the relationship between colposcopic anogenital findings and overall assessment of sexual abuse. DESIGN: Prospective study. SETTING: Regional hospital, Hong Kong. PATIENTS: Seventy-seven children (mean age, 6.5 years; range, 6 months-16 years) referred consecutively for sexual abuse evaluation between July 1999 and June 2002 were included. MAIN OUTCOME MEASURES: Colposcopic anogenital findings (categorised as normal, non-specific, concerning for abuse, or clearly abnormal) were correlated with the overall assessment of likelihood of abuse (classified as no evidence of abuse, possible abuse, probable abuse, or definite abuse). The sensitivity and specificity of clearly abnormal findings in detecting definite abuse were computed, and the diagnostic impact of colposcopy findings were expressed as likelihood ratios. RESULTS: Anogenital findings were normal in 45% of patients, non-specific in 29%, concerning for abuse in 13%, and clearly abnormal in 13%. Seven of the 16 confirmed cases of sexual abuse had normal or non-specific findings. Overall assessment showed that 46% of all patients had no evidence of abuse, 20% had cases of possible abuse, 13% had cases of probable abuse, and 21% had cases of definite abuse. The sensitivity and specificity of abnormal anogenital findings in detecting definite abuse were 56.3% and 98.4%, respectively. Colposcopy showed a fair correlation with the overall assessment of abuse (weighted kappa, 0.245). The diagnostic impact of normal, non-specific, concerning, and clearly abnormal findings in terms of likelihood ratios were 0.23, 1.12, 0.00, and 34.30, respectively. CONCLUSIONS: Anogenital findings are often normal or non-specific in sexual abuse. In general, colposcopy examination findings do not directly reflect the final diagnosis. A category-4 finding on colposcopy is very helpful in confirming definite abuse, whereas other findings do not rule out the diagnosis.

Human infection with influenza H9N2.

We report the clinical features of two cases of human infection with influenza A virus subtype H9N2 in Hong Kong, and show that serum samples from blood donors in Hong Kong had neutralising antibody suggestive of prior infection with influenza H9N2.