Abstract
BackgroundHuman 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 mediumACF. 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 mediumACF.ResultsFor the first one to three passages, cells grown in USSC growth mediumACF 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.ConclusionsThese data suggest that USSC growth mediumACF 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.
Highlights
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
USSCs form spheres in USSC growth mediumACF We have analyzed the growth of USSCs in three serumfree media formulations; HEScGRO, PSM and USSC growth mediumACF
We present a new method to grow USSCs in suspension without losing their multi-lineage differentiation capacity in short term assays
Summary
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. We investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGROTM, PSM and USSC growth mediumACF. Human umbilical cord blood contains a subset of stem cells that can differentiate into cells representative of all three germline layers [1,2,3,4]. Culturing of USSCs depends on growth in medium containing a high concentration of specific batches of fetal calf serum. When cultured in fetal calf serum, USSCs form an adherent monolayer, do not require a feeder layer, and can be cultured without differentiating or losing their proliferative capacity
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