The differentiation capacity of the cells is also maintained after five passages in USSC growth mediumACF(Figures8and9). == Physique 7. can also revert to monolayer growth when cultured on extracellular matrix support (fibronectin or TFR2 Carnosic Acid 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 differentiatein vitroto form cells representative of the three germline layers after five passages. == Conclusions == These data suggest that USSC growth mediumACFmaintains 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. == Background == Human umbilical cord blood contains a subset of stem cells that can differentiate into cells representative of all three germline layers [1-4]. We follow Kgler, the first to describe the multilineage capacity of these cellsin vivo, in calling them “unrestricted somatic stem cells” (USSCs) [1]. A number of studies demonstrate the therapeutic potential of USSCs in bone healing, as accessory cells to reduce graft-versus-host disease, in the repair of myocardial infarcts and as vehicles for gene therapy [1,3,5-8]. Although USSCs are rare compared to haematopoietic stem cells in cord blood, they can be expanded rapidly to yield large numbers of cells for study or transplantation. Culturing of USSCs depends on growth in medium made up of a high concentration of specific batches of fetal calf serum. When cultured in fetal calf serum, USSCs form an Carnosic Acid adherent monolayer, do not require a feeder layer, and can be cultured without differentiating or losing their proliferative capacity. Before USSCs can be used therapeutically, a method for large scale amplification of cells under Good Manufacturing Practice (GMP) conditions is necessary, preferably using defined medium free of xenobiotic components. Fetal calf serum contains undefined factors, which may vary from batch to batch, and that may activate or inhibit stem cell differentiation. The growth of cells in defined medium not only reduces variability, but also eliminates the need for costly and time consuming fetal calf serum batch testing. Additionally, medium that is free of non-human products reduces concerns about transmission of infectious cross-species pathogens (such as prions or zoonosis), and possible immunogenic responses to nonhuman proteins [9-11]. Here we investigate the capacity of three different stem cell culture media HEScGRO, PSM and USSC growth mediumACFto enable maintenance of USSCs in serum-free conditions. PSM is usually of interest as it contains Sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF), factors known to enable the survival and proliferation of human embryonic stem (hES) cells in an undifferentiated state for a prolonged period of time [12,13]. S1P is usually a bioactive sphingolipid present in serum that binds to its own G protein-coupled receptors, sphingosine-1-phosphate receptor 1 to 5 (S1P(1-5)), and activates various intracellular signaling pathways depending on the cell type [14-18]. S1P regulates key biological processes such as cell proliferation, motility, migration and survival in both adult and embryonic stem cell types [13,19-21]. PDGF is usually a growth factor, also present in serum, that is widely described as a potent mitogen [22]. PDGF is comprised of homo- and hetero-dimeric isoforms of polypeptide chains A, B, C and D [23,24]. The response of a cell to a given isoform of PDGF is dependent Carnosic Acid around the signaling pathways activated by the two platelet-derived growth factor receptors (PDGFR- and PDGFR-), and the capacity of the cell to respond to these signals [25-27]. HEScGRO is usually a commercial serum- and animal component-free, defined medium known to support the growth and pluripotency maintenance of hES cells. The media contains FGF2 (basic FGF) that, like PDGF, has been reported as having proliferative and differentiation effects on various cell types [28], and Carnosic Acid figures quite prominently in numerous other adult [29], or embryonic-derived stem cell media formulations [30]. == Results == == USSCs form spheres in USSC growth mediumACF == We have analyzed the growth of USSCs in three serum-free media formulations; HEScGRO, PSM and USSC growth mediumACF. Figures1Aclearly demonstrate Carnosic Acid that cells cultured in HEScGRO and PSM do not grow, whereas cells grown in USSC growth mediumACFproliferate at the same rate as cells grown in stem cell proliferation medium containing 30%.