ABSTRACT
Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method, only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently, we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures, whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry, we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks, indicating that these cells contained rSRA activity. In contrast, CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally, suggesting that the lack of reconstitution was not due to homing defects. In conclusion, we show that after in vitro expansion, rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential, mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells.
Subject(s)
ADP-ribosyl Cyclase 1/deficiency , Antigens, CD34/physiology , Antigens, CD/physiology , Antigens, Differentiation, Myelomonocytic/physiology , Cord Blood Stem Cell Transplantation/methods , Leukocyte Common Antigens/physiology , Severe Combined Immunodeficiency/therapy , Animals , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Pluripotent Stem Cells , Sialic Acid Binding Ig-like Lectin 3ABSTRACT
BACKGROUND: Patient survival in allogeneic cord blood transplantation is critically dependent on total nucleated cell (TNC) count or total CD34+ cell count per kg of body weight. Theoretically, viable CD34+ cell measurement at the time of infusion should give a better indication of the suitability of a certain transplant. The relation between measurements on different samples and viable CD34+ cell count on the graft itself was analyzed. STUDY DESIGN AND METHODS: Viable CD34+ cells were measured with a no-wash, single-platform technique with 7-aminoactinomycin D. Analysis was performed before freezing on the cord blood, after freezing and thawing on the cord blood unit itself, and on various samples. RESULTS: Cord blood volume correlated poorly with viable CD34+ cell content (r=0.24) as did initial TNC count and WBC count (r=0.57 and r=0.48, respectively). In contrast, viable CD34 cell content determined before freezing correlated well with viable CD34 cell content of the graft (r=0.91) but was on average 25 percent higher than after freezing and thawing. The best correlations with the CD34+ cell content of the cord blood unit were obtained with CD34 cell measurements on a separate cryovial (r=0.95). These CD34 cell measurements on frozen samples were found to be very reproducible (r=0.96). CONCLUSION: Viable CD34 cell count of the graft is both accurate and precise when measured on a separate sample frozen together with the cord blood unit. This measurement can be performed by the transplant center to exclude between-laboratory variability.
