A total of 50 human umbilical cord blood (UCB) samples were studied. The hematopoietic stem/progenitor (CD34+) populations were isolated from UCB mononuclear cells (MNC) by means of immunomagnetic separation. Double immunofluorescent staining of UCB CD34+ cells revealed that there was a high proportion (82.33 ± 4.47%) of CD34+ cells co-expressing CD13, while the percentage of CD34+ CD33+ cells was much lower (22.17 ± 3.35%). In contrast, for co-expressing lymphoid differentiation antigens, the proportion of CD34+CD38+ cells (38.34 ± 6.09%) was relatively higher than that of CD34+CD10+ cells (11.52 ± 1.24%) or CD34+CD2+ cells (9.84 ± 2.30%). For stimulating the ex vivo expansion of UCB progenitor cells, no single hematopoietic growth factor (HGF) was efficacious when used alone, while combination of 4 HGFs, such as GM-CSF, G-CSF, IL-3, and SCF could induce a 55-fold increase in the myeloid progenitor cells, day-14 CFU-GM, in a short term of 7 days’ liquid culture. Cryopreservation of UCB as MNC preparations at –196°C could satisfactorily retain the number and activity of CD34+ cells. After thawing, a high recovery rate of about 80% CD34+ cells was obtained. When suspended in liquid cultures containing a combination of 4 HGFs, as shown above, the frozen cord blood progenitor cells could be well expanded, reaching a >50-fold increase in day-14 CFU-GM, which was very similar to that of the fresh UCB samples. In addition, a similar result was also seen in CFU-GEMM, indicating that after cryopreservation the recovered UCB progenitor cells retain an intact clonogeneic ability capable of efficiently responding to hematopoietic growth factors for ex vivo expansion.

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