© Georg Thieme Verlag KG Stuttgart · New York
Phänotypische Unterschiede zwischen CD34 positiven Zellen in verschiedenen Transplantations-MaterialienPhenotypic Differences among "Stem Cells" (SC) from Different Cell Sources Used for Transplantation
13 March 2008 (online)
Transplantations to restore the hematopoietic system were originally performed with cells from the bone marrow (BM) (20) which was considered the only cell source comprising repopulating progenitor cells. The discovery that chemotherapy induced the mobilization of CD34+ cells into the peripheral blood (PB) (14) gave rise to the successful autologous transplantation of PBSC (1, 13). Also cord blood (CB) was found to contain considerable numbers of "stem cells", and to date at least 42 allogeneic transplantations have been performed with this cell source (22, J. Wagner, personal communication). Further investigations led to the successful autologous transplantation of positively selected CD34 + cells from BM and PB (18), and the latest results indicate that it is promising to transplant purified CD34 + cells obtained from cytokine-stimulated donors (4, 10, 15-16). Despite such achievements it remains unclear how many "stem cells" are required per kg of the recipient and how they are phenotypically characterized. In this communication we give examples of typical differences observed by flow cytometry and clonogenic assay between the CD34 + cells contained in the different cell sources. They may explain why it is not sufficient only to analyze the CD34 + cell populations which may represent progenitors of different lineages as well as of various states of differentiation.
CB CD34 + cells are early myeloid progenitor cells with the highest incidence of CFU-mix among the three cell sources. They have a high proliferative potential in vitro. They hardly coexpress B cell antigens and they are partially negative for CD38. Their majority is negative for CD45RA (8). Among the three cell sources, they have the greatest proportion of CD34 +/CDlla- cells. The fact that CD34 + cells coexpress CD33 and HLADR indicates that the latter antigens may not only represent differentiation markers (7). The prolonged period of 25 days post transplantation to reach >500 neutrophils/µl (22) may be due to the early differential state of the progenitors and/or to low CD34 numbers transplanted per kg.
In contrast to CB, the majority of BM CD34 + cells represent relatively committed progenitors as indicated in vitro by a lower cloning efficiency and by high numbers of CFU-G and CFU-M in favour of BFU-E and mainly CFU-mix. Flow cytometry revealed about 80%-90% of the CD34+ cells to coexpress CD45RA. A variable proportion (0% to >70%) of the BM CD34+ MNC may represent B cell progenitors which are positive for CD45RA and which would not support myeloid cell recovery after transplantation.
Chemotherapeutic treatment as well as growth factor stimulation cause the mobilization of CD34 + cells from the BM to the PB. It is noteworthy that B cell progenitors are not, and committed progenitors are hardly mobilized. The PBSC usually represent relatively early myeloid cells the majority of which is negative or dim for CD45RA although they are more differentiated than the CB CD34 + cells.
The observation that CB as well as steady state and cytokine-mobilized PB progenitor cells are never in cell cycling phase (S/G2M) which is in sharp contrast to BM CD34 + MNC. indicates the role of the BM microenvironment for the progenitor cell proliferation (Leitner et al., submitted).
Seit der ersten Beschreibung des CD34-Antigens auf hämatopoetischen Vorläuferzellen vor zehn Jahren (3) wurde eine Vielzahl von Antikörper-Klonen gegen dieses ,,Stammzell'-Antigen auf den Markt gebracht, die größtenteils in der Durchflußzytometrie (am FACS) Verwendung finden. Bei Transplantationen zur Rekonstitution der Hämatopoese liefert diese schnelle Methode wertvolle Information über den relativen und absoluten Gehalt an hämatopoetischen Vorläuferzellen. Die gebräuchlichsten Transplantationsmaterialien sind das Knochenmark und die Zellen des peripheren Blutes, aber auch Nabelschnurblut wird zunehmend verwendet. Wir konnten zeigen, daß die in diesen Materialien enthaltenen CD34-positiven Zellen meist unterschiedliche, für das jeweilige Material typische Subpopulationen darstellen. Unsere Befunde zeigen, daß es nicht genügt, mit der reinen Einfarben-Analyse die Zahl der CD34-positiven Zellen zu bestimmen, sondern daß die phänotypische Charakterisierung der ,,Stammzell"- Subpopulationen mittels Zwei- und Dreifarben-Analyse eine wesentlich verbesserte Aussage über die im Transplantat enthaltenen Progenitorzellen ermöglicht.