At the end of culture, cells on ECM were confluent, fibroblast-like, and spindle-shaped (Fig. 4a). UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. == Conclusions == These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications. Keywords: Umbilical cord blood, CD146-positive cells, Plastic non-adherent cells, Extracellular matrix-adherent cells, Pluripotent stem cells, Extracellular matrix, Three germ layer tissue formation in vivo == Background == A practical and reliable source of pluripotent Rabbit Polyclonal to ATP1alpha1 and/or multipotent stem cells holds the key to future stem cell therapies. Despite the great potential shown by human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), there are a number of challenges that need to be overcome before they can be widely endorsed for use in large numbers of patients suffering from various aged-related diseases. These challenges include ethical concerns with regard to hES cells, potential long-term safety of transplanted iPSCs, and the considerable level of technical skill and cost associated with preparing both types of cells. Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs) but also non-hematopoietic stem cells (NHSCs) that can differentiate into many distinct cell types including osteoblasts, chondrocytes, myocytes, endothelial cells, and neurons [1, 2]. Based on these findings, UCB has been proposed as an alternative source of mesenchymal stem cells (MSCs) for stem cell-based therapies [3, 4]. Compared to bone marrow (BM), a rich source of MSCs, UCB is widely available in abundance and can be harvested with little harm (i. e., low morbidity/mortality) to the donor [5, 6]. However , most studies report that the frequency of NHSCs in UCB AT7867 2HCl is extremely low (~5 to 350 out of 109mononuclear cells (MNCs)) [69] and, compared to BM-derived MSCs (BM-MSCs), these cells are very difficult to grow in standard culture systems AT7867 2HCl [1012]. As a result, progress in developing UCB as a source of NHSCs for stem cell-based therapies has been largely thwarted. Unlike HSCs, there are no reliable cell surface markers that specifically define MSCs or NHSCs. As a result, these cells are routinely obtained from BM and other tissues (e. g., UCB) using a standardized method based on adhesion to tissue culture plastic (TCP) [8, 1315]. However , there is evidence that the non-adherent cell population contains a significant number of MSCs and the behavior of these cells is very different from those that adhere to TCP [1618]. Based on these observations, we AT7867 2HCl hypothesized that UCB contains an extremely immature population of NHSCs that have been previously overlooked because they fail to attach to TCP. Previously, we reported on the production and characterization of a native extracellular matrix (ECM) generated by BM stromal cells [19, 20]. This ECM, deposited onto TCP dishes by the cells AT7867 2HCl and subsequently decellularized, is 80100 m thick and displays a unique tissue-like architecture. It consists of more than 70 different matrix proteins including collagens, fibronectin, small leucine-rich proteoglycans, and several basement membrane components [19, 20]. MSCs cultured on this BM-derived ECM (BM-ECM) exhibit enhanced attachment, proliferation, and retention of stem cell properties, including differentiation into multiple cell lineages as well as the capacity for skeletogenesis [1921]. The current study tests the above hypothesis and determines whether BM-ECM facilitates the efficient isolation of UCB-NHSCs by enhancing attachment and proliferation and promoting the retention of their stem cell properties. == Methods == == Isolation and culture of NHSCs from human UCB == UCB was purchased from South Texas Blood & Tissue Center (San Antonio, TX, USA). MNCs were isolated from UCB using Ficoll-Paque Premium density media (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) as described previously [14]. MNCs were suspended in growth media containing alpha-minimal essential media (-MEM; Life Technologies, Grand Island, NY, USA), penicillin (100 U/ml), streptomycin (100 g/ml; Biofluids, Rockville, MD, USA), 15% pre-selected fetal bovine serum.