Lysdahl Helle, Gabrielsen Anette, Minger Stephen L, Patel Minal J, Fink Trine, Petersen Karsten, Ebbesen Peter, Zachar Vladimir
Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg Oest, Denmark.
Reprod Biomed Online. 2006 Jan;12(1):119-26. doi: 10.1016/s1472-6483(10)60990-x.
In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1-60, TRA-1-81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark.
2003年9月，丹麦批准的一项立法使利用体外受精产生的多余人类胚胎进行临床用途的研究合法化，以建立人类胚胎干细胞（ES）培养物。本研究的目的就是建立这样的干细胞系。新鲜的多余胚胎在获得捐赠者的知情同意后被捐赠出来。胚胎被培养成囊胚，然后采用免疫外科手术方法，分离出内细胞团，并将其接种于经辐照的人包皮成纤维细胞上，在添加了敲除血清替代物、碱性成纤维细胞生长因子（bFGF）和白血病抑制因子（LIF）的敲除型 Dulbecco改良 Eagle培养基（KnockOut D-MEM）中进行培养。在12个月的时间里，共捐赠了198个胚胎。4个分离出的内细胞团发育成了假定的ES细胞系，即CLS1、CLS2、CLS3和CLS4，目前这些细胞系已连续培养了8个月，相当于传代30次。这些细胞表达了未分化人类ES细胞的标志物：阶段特异性胚胎抗原-4、肿瘤相关抗原（TRA）-1-60、TRA-1-81、八聚体结合转录因子4（OCT4）、Nanog基因、性别决定区Y框蛋白2（SOX2）和成纤维细胞生长因子4（FGF4）。这些细胞表现出高水平的端粒酶活性，具有正常的核型，并且已成功冻存和解冻。最后，这些细胞在体外显示出分化为源自所有三个胚层的细胞类型的潜力。据认为，本研究中描述的细胞系是丹麦建立的首批人类ES细胞。
