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In Vitro Expanded Cells Contributing to Rapid Severe Combined Immunodeficient Repopulation Activity Are CD34+38−33+90+45RA−

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.