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First published online September 14, 2006
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2006-0256v1
25/1/107    most recent
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Submitted on April 25, 2006
Accepted on September 1, 2006

Tissue-Specific Stem Cells

IN VITRO EXPANDED CELLS CONTRIBUTING TO RAPID SCID REPOPULATION ACTIVITY ARE CD34+38-33+90+45RA-

Katrien Vanheusden 1, Stefanie Van Coppernolle 2, Magda De Smedt 1, Jean Plum 1, Bart Vandekerckhove 1*

1 Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Ghent University Hospital, Ghent, Belgium
2 Dienst voor het bloed, Rode Kruis - Vlaanderen, Ghent, Belgium

* To whom correspondence should be addressed. E-mail: Bart.Vandekerckhove{at}ugent.be.


  Abstract

Expansion of hematopoietic stem cells (HSC) could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid 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 4-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/NOD 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 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.

Key Words. Hematopoiesis, Stem cell, Stem cell culture, NOD/SCID mice




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