Correspondence 947 7 Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobaya- 8 Taki T, Ida K, Bessho F, Hanada R, Kikuchi A, Yamamoto K, Sako shi M, Bessho F, Yanagisawa M, Hayashi Y. Tandem duplication M, Tsuchida M, Seto M, Ueda R, Hayashi Y. Frequency and clini- of the FLT3 is found in acute lymphoblastic as well cal significance of the MLL gene rearrangements in infant acute as but not in myelodysplastic syndrome leukemia. Leukemia 1996; 10: 1303–1317. or juvenile chronic myelogenous leukaemia in children. Br J Haematol 1999; 105: 155–162.

Loss of CD38 antigen on CD341CD381 cells during short-term culture

TO THE EDITOR Table 1 Antigens on CD34+ subpopulations after short-term culture of purified cord CD34+CD38+ cells

The earliest progenitor in the hierarchy of hematolymphopoietic cells, Population Myeloid Erythroid Dendritic the hematopoietic (HSC), can repopulate the Marker CD33 Glycoph. CD1a CD10 CD3 after transplantation and sustain hematopoiesis, generating all known CD14 A CD80 CD19 CD4 mature blood cells. Assay systems to measure repopulating capacity CD16 CD86 CD20 CD8 of HSCs in humans are not available. However, early progenitors can be detected by their ability to produce cobble stone areas (CAFC), CD34+CD38+ 55/45a 11/3 9/6 2/1 3/5 and colony-forming cells (CFC) for at least 5 weeks on stroma (LTC- CD34+CD38− 54/30 8/2 ,1/,1 ,1/,1 ,1/,1 IC). In addition, the repopulating capacity of human HSCs can be measured in mice with severe combined immunodeficiency (SRC). aThe percentage of cells positive for the markers within the given Several surface antigens such as CD34, CD38, and HLA DR have population for two independent experiments is shown. been used as markers to enrich HSC from different sources.1 However, even the most highly enriched populations still remain heterogeneous. A more than 1000-fold enrichment of SRC and LTC-IC was found in CD34+CD38− cells compared with unseparated mononuclear cells 2,3 from cord blood or bone marrow. Here, we studied whether after + + cluded that CD34 CD38 cells lost the CD38 antigen during culture ex vivo culture CD38 still remains a marker for more differentiated + while remaining CD34 . progenitor cells. + We then determined whether the initial staining of cells for FACS Cord blood CD34 hematopoietic progenitor cells were purified by sorting with anti-CD38 directly after isolation interfered with CD38 the MACS progenitor isolation (Miltenyi Biotec, Bergish Gladbach, antigen detection on day 4. Directly after MACS purification, half the Germany) and cultured for 4 days. The effect of two different growth cells were stained with anti-CD38 conjugated with fluorescein and factor combinations on the expression of CD34 and CD38 was ana- then cultured for 4 days with FL, IL-3 and SCF. The remaining cells lyzed. Cells were either cultured in BIT medium supplemented with were cultured directly as a control. On day 4, both populations Flt-3 ligand (FL, 100 ng/ml), stem cell factor (SCF, 100 ng/ml), and β (prestained and directly cultured) were stained with anti-CD38 interleukin-3 (IL-3, 20 ng/ml, or with IL-1 (3 ng/ml), SCF (10 ng/ml), cychrome and analyzed by flow cytometry. No difference in the inten- and Il-3 (20 ng/ml, were obtained from TeBu, Frankfurt sity of anti-CD38 staining was seen (data not shown). a.M., Germany). These conditions were previously shown to expand + − + 4,5 Finally, we tested whether the CD34 CD38 cells that arose after LTC-IC and CD34 cells, respectively. On day 4, cells were stained culture were enriched for cells of specific lineages. CD34+CD38+ cells with anti-CD34 and anti-CD38 (antibodies were obtained from were sorted, cultured for 4 days and then stained with CD34, CD38, Pharmingen, San Diego, CA, USA) and analyzed on a FACScalibur and with antibodies against specific lineage antigens. The results of (Becton Dickinson, Heidelberg, Germany). Before culture, 95% of the + + the three-color flow cytometric analysis are shown in Table 1. On cells were CD34 CD38 (Figure 1a). After culture in SCF, IL-3 and IL- + − + CD34 CD38 cells no significant staining was seen with antibodies 6, most cells still remained CD38 and 21% had lost the CD34 antigen against B cell, T cell, or dendritic markers. A low percentage (Figure 1c). In contrast, cells cultured in FL, SCF and IL-3 mostly + − expressed Glycophorin-A and myeloid antigens (CD33, CD14, CD16) retained the CD34 antigen and 31% had become CD34 CD38 were found on approximately half of the day 4 CD34+CD38− cells. (Figure 1b). This effect could be reproduced with bone marrow and These results show that although within the CD34+CD38− cells there peripheral blood progenitor cells (data not shown). is a population with myeloid potential, a considerable number of cells + − The day 4 CD34 CD38 cells could have arisen by massive expan- is negative for standard lineage markers. + − sion of CD34 CD38 cells during culture or by loss of the CD38 In conclusion, the CD34+CD38− Lin− cells that arose from a + marker on cells that were initially CD38 (Figure 1a). Therefore, CD34+CD38+ population during culture have a phenotype normally + + CD34 CD38 cells were purified by MACS and subsequent cell sort- found on early hematopoietic progenitors directly after isolation. ing (MoFlo sorter; Cytomation, Fort Collins, CO, USA; Figure 1d) and However, functional studies, such as LTC-IC and SRC assays will be then cultured for 4 days in BIT with FL, IL-3 and SCF. On day 4, cell necessary to clarify whether this population is indeed enriched in numbers had increased by three-fold with a viability of over 80% and HSC, or whether the CD38 antigen is not a reliable indicator of differ- 32% of the cells were CD34+CD38− (Figure 1e). We therefore con- entiation after ex vivo culture.

Correspondence: D von Laer, Heinrich-Pette-Institut, Martinistr. 52, D-20251, Hamburg, Germany; Fax: 49-40-48051187 Received 29 November 1999; accepted 15 December 1999

Leukemia Correspondence 948

Figure 1 CD34+CD38+ cells develop into CD34+CD38− cells during culture. MACS-purified CD34+ cord blood cells were stained before (Day 0, a) and after 4 days of culture (Day 4) with anti-CD34 and anti-CD38. Culture medium was supplemented with FL, SCF, and IL-3 (b) or with IL1β, IL-3 and SCF (c). In addition, CD34+CD38+ cells were purified by MACS and subsequent cell sorting (Day 0, d), cultured with IL3, SCF and FL and then stained with anti-CD34 and anti-CD38 (day 4, e).

Acknowledgements 2 Larochelle A, Vormoor J, Hanenberg H, Wang JCY, Bhatia M, Lap- idot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick This work was supported by Bundesministerium fu¨r Bildung und For- JE. Identification of primitive human hematopoietic cells capable schung grant 01KV9811.4 of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nature Med 1996; 2: 1329–1337. 3 Reems JA, Torok-Storb B. Cell cycle and functional differences between CD34+/CD38hi and CD34+/38lo human marrow cells after in vitro exposure. Blood 1995; 85: 1480–1487. D von Laer11Heinrich-Pette-Institut fu¨r Experimentelle Virologie 4 Petzer AL, Zandstra PW, Piret JM, Eaves CJ. Differential cytokine A Corovic1 und Immunologie effects on primitive (CD34+CD38−) human hematopoietic cells: B Vogt1 an der Universita¨t Hamburg novel responses to Flt3-ligand and thrombopoietin. J Exp Med B Fehse22Zentrum fu¨r Knochenmarktransplantation, 1996; 183: 2551–2558. S Roscher1 Universita¨tskrankenhaus Eppendorf 5 Fehse B, Uhde A, Fehse N, Eckert HG, Clausen J, Ruger R, Koch A Rimek33CELLTEC GmbH, Hamburg, Germany S, Ostertag W, Zander AR, Stockschlader M. Selective immunoaf- C Baum1 finity-based enrichment of CD34+ cells transduced with retroviral W Ostertag1 vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene. Hum Gene Ther 1997; 8: 1815–1824. Reference

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