McGuckin C P, Forraz N, Baradez M-O, Navran S, Zhao J, Urban R, Tilton R, Denner L
Stem Cell Therapy Programme, School of Life Sciences, Kingston University, Kingston upon Thames, Surrey, UK.
Cell Prolif. 2005 Aug;38(4):245-55. doi: 10.1111/j.1365-2184.2005.00346.x.
When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.
胚胎干细胞何时能应用于临床？答案很简单——短期内不行！要生产大量用于移植的同质组织，且无需饲养层，并具有合适的受体免疫表型，这是一个重大的科学障碍，尽管成体干细胞提供了一种更符合伦理道德的替代来源。全球每年一亿的人口出生率表明脐带血是最大的未被开发的干细胞来源，其具有原始免疫状态和相对未缩短的端粒长度等优势。在此，我们报告了世界上首次可重复生产表达胚胎干细胞标志物的细胞——脐带血来源的胚胎样干细胞（CBEs）。通过剖宫产选择性分娩后获取的脐带血，经过连续免疫磁珠法去除有核粒细胞、红细胞和造血髓系/淋巴系祖细胞。在微量培养瓶中进行7天的高密度培养（10⁵细胞/毫升，IMDM培养基，10%胎牛血清，10纳克/毫升血小板生成素，50纳克/毫升flt3配体，20纳克/毫升c-kit配体）。CBE集落形成并附着于基质；这些集落维持培养6周，然后传代培养并至少持续培养13周。CBEs对TRA-1-60、TRA-1-81、SSEA-4、SSEA-3和Oct-4呈阳性，但对SSEA-1呈阴性，这表明其在人类干细胞范畴内受到限制。CBEs还在微重力生物反应器中用肝细胞生长培养基（IMDM培养基，10%胎牛血清，20纳克/毫升肝细胞生长因子，10纳克/毫升碱性成纤维细胞生长因子，10纳克/毫升表皮生长因子，10纳克/毫升c-kit配体）进行培养。4周后发现这些细胞表达特征性的肝脏标志物，细胞角蛋白-18、甲胎蛋白和白蛋白。因此，此类CBEs是胚胎干细胞在干细胞研究方面可行的人类替代物，不受伦理限制且具有临床应用潜力。
