Department of Paediatrics, The University of Melbourne, Royal Children's Hospital, Victoria, 3052, Australia.
BMC Biotechnol. 2009 Dec 15;9:101. doi: 10.1186/1472-6750-9-101.
Human umbilical cord blood-derived unrestricted somatic stem cells (USSCs), which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. A major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. In this study, we investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGRO, PSM and USSC growth medium ACF. Our findings demonstrate that USSCs do not grow in HEScGRO or PSM, but we were able to isolate, proliferate and maintain multipotency of three USSC lines in USSC growth medium ACF.
For the first one to three passages, cells grown in USSC growth medium ACF proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. Upon dissociation of spheres, cells continue to grow in suspension and form new spheres. Dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support (fibronectin or gelatin), or in medium containing fetal calf serum. Analysis of markers associated with pluripotency (Oct4 and Sox2) and differentiation (FoxA2, Brachyury, Goosecoid, Nestin, Pax6, Gata6 and Cytokeratin 8) confirms that cells in the spheres maintain their gene expression profile. The cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages.
These data suggest that USSC growth medium ACF maintains USSCs in an undifferentiated state and supports growth in suspension. This is the first demonstration that USSCs can grow in a serum- and animal component-free medium and that USSCs can form spheres.
人脐带血来源的无限制体干细胞（USSCs）具有多能性分化能力，目前正在研究用于多种治疗应用。其临床应用的一个主要障碍是，体外扩增仍然依赖于胎牛血清，而胎牛血清可能是病原体的来源。在这项研究中，我们研究了三种不同的干细胞培养基在无血清条件下支持 USSCs 的能力；HEScGRO、PSM 和 USSC 生长培养基 ACF。我们的研究结果表明，USSCs 不能在 HEScGRO 或 PSM 中生长，但我们能够在 USSC 生长培养基 ACF 中分离、增殖和维持三种 USSC 系的多能性。
在前一到三代，在 USSC 生长培养基 ACF 中生长的细胞增殖并保持其形态，但随着传代的进行，细胞形成球形细胞聚集体。当球体解离时，细胞继续在悬浮液中生长并形成新的球体。当在细胞外基质支持物（纤连蛋白或明胶）上或在含有胎牛血清的培养基中培养时，解离细胞也可以恢复为单层生长。与多能性相关的标记物（Oct4 和 Sox2）和分化（FoxA2、Brachyury、Goosecoid、Nestin、Pax6、Gata6 和 Cytokeratin 8）的分析证实，球体中的细胞保持其基因表达谱。球体中的细胞还保留在体外分化形成代表三个胚层的细胞的能力，经过五代传代后。
这些数据表明，USSC 生长培养基 ACF 使 USSCs 保持未分化状态，并支持悬浮生长。这是首次证明 USSCs 可以在无血清和动物成分的培养基中生长，并且 USSCs 可以形成球体。
