Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.

Chondrogenesis from umbilical cord blood cells stimulated with BMP-2 and BMP-6.

Umbilical cord blood contains undifferentiated mesenchymal stem cells (MSCs) with chondrogenic potential that may be used for the repair of joint damage. The role of growth factors during the process of chondrogenesis is still not entirely understood. The objective of this study was to evaluate the formation of chondrocytes, cartilaginous matrix and type II collagen from human umbilical cord blood stem cells exposed to two different growth factors, BMP-6 and BMP-2, while being cultured as a micromass or a monolayer. Umbilical cord blood was obtained from full-term deliveries, and then, mononuclear cells were separated and cultured for expansion. Afterward, these cells were evaluated by flow cytometry using antibodies specific for MSCs and induced to chondrogenic differentiation in micromass and monolayer cultures supplemented with BMP-2 and BMP-6. Cellular phenotype was evaluated after 7, 14 and 21 days by RT-PCR and Western blot analysis to identify the type II collagen and aggrecan. The expanded cells displayed surface antigens characteristic of mesenchymal progenitor cells and were negative for hematopoietic differentiation antigens. Type II collagen and aggrecan mRNAs were expressed from day 14 in cells stimulated with BMP-2 or BMP-6. Type II collagen was demonstrated by Western blotting in both groups, and the greatest expression was observed 21 days after the cells were stimulated with BMP-2 cultured in micromass. BMP-2 in micromass culture was more efficient to induce the chondrogenesis.

Early proliferation of umbilical cord blood cells from premature neonates.

BACKGROUND AND OBJECTIVE: Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates. MATERIAL AND METHODS: Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting. RESULTS: Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells. CONCLUSION: These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.