Cells at passages 4 or 5 5, or when the expression of the haematopoietic markers CD45, CD34 and CD14 was 1.5%, were seeded in 25 cm2tissue culture flasks. were preserved throughout long-term culture. Even at passage 24 cells remained Nestin+, CD133+and >95% were positive for CD105, CD73, CD29 KRT4 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. Asenapine == Conclusions == This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice. == Introduction == In recent years, mesenchymal stem cells (MSC) received considerable attention as a potential source of cell-based therapies and as a cell type that supports the engraftment of haematopoietic stem cells (HSC)[1],[2]. The usual source of MSC is the bone marrow (BM), which is not easy to obtain from healthy donors as well as umbilical cord blood (UCB). The advantages of UCB as the source of MSC are the availability of units[3],[4]and the primitive nature of UCB-derived MSC[5],[6],[7]. BM- and UCB-derived MSC are presumably highly similar precursors as they share the following features: (i) capacity of self-renewal[8], (ii) multipotency, allowingin vitrodifferentiation into mesenchymal tissues (bone, cartilage, tendon, muscle, adipose tissue, stroma) and possibly non-mesenchymal tissues (neuronal, endothelial and hepatic)[9],[10],[11], (iii) formation of colonies of fibroblastic cells (CFU-F)[12], (iv) expression of MSC markers (CD29, CD44, CD73, CD105) and lack of haematopoietic markers (CD14, CD34, CD45)[4],[13]and (v) migration to inflammatory sites, stimulation of proliferation/differentiation of resident progenitor cells and promotion of recovery of injured cells through growth factor secretion and matrix remodeling[14],[15],[16]. Although the frequency of MSC referred Asenapine here as undifferentiated cells is much higher in BM (0.0010.1%) than in UCB (0.00003%) and some reports have even doubted the presence of MSC in UCB[17],[10],[4],[18],[19], UCB-derived MSC have a better potential to expand and can give rise to up to 1015cells[17],[4]. However, the scarcity of MSC in UCB and the lack of a robust protocol to reproducibly expand MSC from UCB units have hampered clinical applications[17],[4],[20],[19]. It can not be excluded that the low numbers of MSC in cord blood actually derive from placental MSC that were released into cord blood due to mechanical stress during UCB isolation procedure. Several studies reported that MSC can be isolated and established from only 2063% of the cord blood units[3],[8],[21], questioning the feasibility of MSC isolation and cultivation from UCB. Here, we describe a novel, simple and reproducible method, which is based on stroma-free liquid culture, to expand substantial numbers of multipotent MSC from only a small number of UCB-derived mononuclear cells (MNC). This method allows an extensive expansion of non-adherent HSC plus a marked increase in adherent MSC. MSC producedin vitroby this novel culture method maintain their stem cell properties of self-renewal and multi-lineage differentiation for a long-time (up to passage 24), even following cryopreservation. == Methods == == Ethics Statement == All experimental work presented in this study has been approved by the local institutional review board. == Umbilical Asenapine cord blood sample collection and cell processing == UCB from full-term deliveries was.