BACKGROUND: Differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro has been studied in great detail in the world. However, much of what is currently known about cardiomyocyte differentiation from ES cells has been learned from studies on mouse in China, few studies are on human ES cells.OB JECTIVE: To investigate the differentiation effcacy of human ES cells into functional cardiomycytes with the human H14 ES cell line.DESIGN: Suspending method was used to form pseudo-embryo proper of human ES cells so as to observe ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phases.SETTING: Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong.MATERIALS: Human ES cell line H14 was obtained from WiCell Research Institute (Wisconsin, USA) with a license agreement.METHODS: The experiments were carried out in the Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong from August to December of 2006.The H14 ES cell colony was used to form embryoid bodies (EBs) by using suspending method. Four days later,pseudo-embryo proper was cultured in gelatin-coating 6-well culture plate (5-10 embryo proper/well) and spontaneously differentiated into moving pseudo-embryo proper. Rhythmic contraction was observed under microscope and RT-PCR was used to detect expression of special genes of myocardium.MAIN OUTCOME MEASURES: Ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phasesRESULTS: Spontaneously contracting cells appeared as cluster and were identified in approximately 2% of EBs at differentiation day 8 and increased to as many as 10% of the EBs by day 16. The beating rate of contracting cells arranged at 70-100 beats per minute. RT-PCR analyses demonstrated that cells isolated from spontaneous beating areas within the EB expressed the cardiac transcription factors GATA-4 and Nkx2.5, cardiac progenitor gene Isl-1 and cardiomyocyte marker gene α-MHC.CONCLUSION: This is the first time to report human ES cells can effectively differentiate into functional cardiomyocytes in China.

Objective To investigate the effect of miR-15b and miR-16 on the expression of vascular endothelial growth factor (VEGF) protein in human embryonic lung fibroblast (HLF) cells under hyperoxia.Methods The expression level of miR-15b and miR-16 was up-regulated and down-regulated in HLF cells by transfection technology, respectively.The expression of VEGF protein in HLF cells was assessed by Western blot.Furthermore, under hyperoxia exposure in vitro, the expression of miR-15b, miR-16 and VEGF protein in HLF cells was analyzed.Results Up-regulation of miR-15b and miR-16 suppressed VEGF protein expression, while down-regulated miR-15b and miR-16 promoted VEGF protein expression.In addition, hyperoxia exposure induced up-regulation of miR-15b and miR-16, but down-regulation of VEGF protein in HLF cells.Conclusions Hyperoxia exposure may up-regulate the expression level of miR-15b and miR-16, but suppress VEGF protein expression.These may contribute to the development of bronchopulmonary dysplasia.

Objective To investigate the expression and clinical significance of miR-125b in patients before and after treatment of acute myeloid leukemia (AML) with different types.Methods The levels of miR-125b were measured by using real-time fluorescence quantification PCR (qRT-PCR) in 65 AML patients (all AML samples from the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University,and the control samples isolated from cord blood which were obtained after normal full-term delivery of babies) before and after treatment,and then the relationship between the levels of miR-125b and patients＇ sex,age,peripheral blood cells,type clinically,relapse and therapeutic effect were analyzed.Results Of newly diagnosed AML patients and the control samples,miR-125b positive rate was 100％.The miR-125b expression levels in the control group was 1.50,and its expression in AML was 11.06,and the difference was significant (P =0.036 6).In complete remission (CR) group of acute promyelocytic leukemia (M3),the expression of miR-125b after induction therapy was significantly reduced,and CR rate of miR-125b decreased group was 91.3％,while that of the increased group was 50.0％,and the difference was significant (P =0.042 6).The miR-125 b expression level was decreased from 29.7 ± 4.9 to 19.2 ± 6.0 after chemotherapy,and the difference was significant (P ＜ 0.036 6).In non-M3 AML,CR rate of miR-125 b decreased group was 86.7 ％,while that of the increase group was 42.1％,and the difference was significant (P =0.021 5).There was no correlation between miR-125b expression and patients＇ gender,age and peripheral blood cells.Conclusions The differences in expression of miR-125b is very important in disease occurrence and progress.Using qRT-PCR to dynamically detect the expression of miR-125b dynamically in AML patients before and after therapy may predict outcome more precisely and has the potentials as an effective biomarker in determining prognosis,monitoring the risk of recurrence,and guiding the treatment.