title: Assessment of cell viability and apoptosis in human umbilical cord blood following storage.
abstract: There is increasing use of human umbilical cord blood (UCB) stem cells for unrelated donor transplantation. Successful engraftment depends in large measure on the dose and quality of cells in the UCB unit. In the present study, we attempted to identify a simple and rapid technique for assessing the quality and recovery of UCB cells following laboratory manipulation. Mononuclear cells (MNC) from fresh (<48 h old) and thawed UCB units were stained with 7-amino-actinomycin D (7-AAD), revealing 2-3% dead cells. The frequencies of apoptotic cells in fresh and thawed sample were similar. However, UCB held for 72 h showed higher levels of cell deterioration. Interestingly, staining with 7-AAD was more sensitive to cellular damage than was uptake of Trypan Blue. 7-AAD staining of MNC also correlated with retention of hematopoietic function (progenitor assays) such that 7-AAD staining frequencies <20% predicted maintenance of hematopoietic cells. Importantly, hematopoietic precursors were less susceptible to storage injury than were UCB MNC as a whole. MNC showed higher levels of 7-AAD staining and apoptosis than did CD34(+) cells. This observation was confirmed in studies of caspase-3 activation, where MNC consistently showed higher frequencies of activation than did CD34(+) cells, especially after overnight storage. Furthermore, antiapoptotic proteins Bcl-2 and Bcl-x were expressed more consistently in CD34(+) cells than in total MNC. In contrast, Bax levels increased with MNC apoptosis. In conclusion, the data suggest that 7-AAD staining of UCB MNC provides a rapid and simple technique for assessing the viability, recovery, and hematopoietic functionality of stored UCB.
authors.affiliation: Pacific Northwest Regional Blood Services, American Red Cross, Portland, OR 97208, USA. mx4r@cms.mail.virginia.edu
authors.forename: Mang
authors.initials: M
authors.lastname: Xiao
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Dooley
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis; D017209:Apoptosis; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D015925:Cryopreservation / Q000379:methods; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D010641:Phenotype; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013048:Specimen Handling; D013234:Stem Cells; D013997:Time Factors; D014343:Trypan Blue / Q000494:pharmacology; D051020:bcl-X Protein
chemical_list: D018952:Antigens, CD34; C494811:BCL2L1 protein, human; D004396:Coloring Agents; D005456:Fluorescent Dyes; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D014343:Trypan Blue