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LETTER

In Vitro Identification of a Cord Blood CD133⁺CD34^–Lin⁺ Cell Subset that Gives Rise to Myeloid Dendritic Precursors

^a Stem Cell Transplant Unit, "Aghia Sophia" Children’s Hospital, Thivon and Levadias, Athens, Greece;
^b Immunology Department and National Histocompatibility Center, "G. Genimmatas" General District Hospital, Athens, Greece;
^c Department of Immunobiology, General District Maternity Hospital "Helena Venizelou," Athens, Greece

Correspondence: Evgenios Goussetis, M.D., BMT-Unit, "Aghia Sophia" Children’s Hospital, Thivon and Levadias, 11527 Athens, Greece. Telephone: (30)-210-7467303; Fax: (30)-210-7778822; e-mail: bmtlab{at}paidon-agiasofia.gr

Received June 24, 2005; accepted for publication November 22, 2005.

	INTRODUCTION

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Using the novel stem cell marker CD133, a functional hierarchy within the CD34⁺ cell population has been described, indicating that CD133⁺CD34⁺ cells are enriched in primitive and myeloid progenitors, whereas CD133^–CD34⁺ cells are committed mainly to the B-cell and erythroid lineages [1, 2]. Interestingly, among CD133⁺ cells, a small cell subset has been found that did not coexpress CD34 or any other lineage-specific marker. Further work on these cells has shown that CD133⁺CD34^–Lin^– cells are the most primitive blood cells identified to date [3]. Of note, a CD133⁺CD34^–Lin⁺ cell subset in fresh bone marrow, peripheral blood, or cord blood (CB) has never been detected, in contrast to the CD133^–CD34⁺Lin⁺ subset that comprises about 30% of the total CD34⁺ cell population [2]. In expansion cultures, however, it has already been observed that CD133⁺CD34⁺ cells generate both CD133^–CD34⁺ and CD133⁺CD34^– cells [4–7]. The latter subset’s identity as a biologically distinct stage in the hematopoietic cell hierarchy has not been further investigated to date. We isolated CB CD133⁺ cells (n = 12) by using the MACS-AC133 Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany, http://www.miltenyibiotech.com) and subsequently cultured them in StemSpan medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) with two different cytokine (R&D Systems, Abingdon, Oxon, U.K., http://www.rndsystems.com) combinations: (a) FTS6' (300 ng/ml Flt-3 ligand [FL], 50 ng/ml thrombopoietin [TPO], 100 ng/ml stem cell factor [SCF], and 25 ng/ml interleukin-6 [IL-6]) and (b) TS6' (50 ng/ml TPO, 100 ng/ml SCF, and 25 ng/ml IL-6). In cultures supplemented with FTS6, the emergence of a CD133⁺CD34^– subset was observed. Starting from 2 x 10⁴ CD133⁺ cells on day 0, total cell recovery on days 7, 14, and 21 was 1.2 ± 0.25 x 10⁵, 0.8 ± 0.4 x 10⁶, and 1 ± 0.2 x 10⁷, respectively. By days 7, 14, and 21 of culture, CD133⁺ CD34^– cells represented 16% ± 3.2%, 16.4% ± 4.8%, and 15.5% ± 5.2% of total nucleated cells, respectively, whereas CD133^–CD34⁺ cells were found to be 4.2% ± 1.4%, 3.6% ± 0.5%, and 1.2% ± 0.8% of total nucleated cells, respectively. On the contrary, cytokine combination TS6 was shown to induce differentiation of CD133⁺CD34⁺ cells throughCD133^–CD34⁺ progenitors(Fig.1A). SortedCD133⁺ CD34^– cells were positive for CD13 and CD33 antigens (Fig. 2A) but negative for other stem cell markers (CD117 and CD90). Day-7 CD133⁺CD34^–Lin⁺ cells demonstrated a proliferative response by increasing the CD133⁺CD34^–Lin⁺ cell number and the total cell number by 12 ± 4.2– and 68.4 ± 6.8 –fold after 2 weeks, respectively. In differentiation cultures, whereas the CD133⁺CD34⁺ subset maintained the capacity to generate erythroid, megakaryocytic, monocytic, and dendritic cells (DCs), CD133⁺CD34^– and CD133^– CD34⁺ cells demonstrated commitment to monocytic-dendritic and erythroid lineages, respectively. Under DC culture conditions (FL, GM-CSF, and IL-4), 1 x 10⁴ CD133⁺CD34^–Lin⁺ cells resulted in a recovery of 2.6 ± 0.8 x 10⁵ cells after 2 weeks. Immunophenotypic analysis of these cells revealed CD1a⁺CD11c⁺DR⁺ cells at a percentage of 32.2% ± 4.5%, CD1a^–CD11c⁺DR⁺ cells at 20.6% ± 6%, and CD1a⁺CD11c^–DR⁺ cells at 18.4% ± 4.8% (Fig. 2B). Further phenotypic analysis of these cells showed that they expressed the co-stimulatory molecules CD86, CD80, CD83, and CD40; CD14 was present in 20.5% ± 4.8% of cells, but its expression was dim. All cells were CD33⁺, but B-cell, T-cell, and natural killer cell markers (CD19, CD3, and CD56) were not detected. Cytospins showed less rounded cells with hyperlobulated nuclei that did not stain for myeloperoxidase or specific esterase and were predominantly negative for nonspecific esterase. These phenotypic, morphologic, and cytochemical characteristics indicate that myeloid dendritic precursors developed from CD133⁺CD34^–Lin⁺ cells under culture conditions favoring DCs [8, 9]. CD133⁺CD34^– cells from four different CB samples were plated in a series of limiting cell doses into 96-well plates (0, 1, 3, 10, 30, and 100 cells per well and one plate per dilution). Cultures supplemented with FL, GM-CSF, IL-4, and tumor necrosis factor– were carried for 2 weeks, and wells that did not contain more than 20 viable cells at the end of 2 weeks were scored as negative. DPs were identified by fluorescence-activated cell sorting after labeling with CD1a and CD11c. The cell dilution that results in 37% negative wells is the proportion to be expected from the Poisson distribution when there is on average one progenitor cell per well. Linear regression analysis of negative wells at each cell concentration revealed a DC progenitor frequency of 1:14 (Fig. 2C). In previous studies, the presence of a CD133⁺CD34^– subset in expansion cultures was considered either a result of aberrant antigen expression due to the culture conditions or an accumulation of a rare stage of hematopoietic hierarchy probably corresponding to the recently described multipotent CD133⁺ CD34^–Lin^– subset [4, 5]. Our findings clearly demonstrate that CD133⁺CD34^– cells generated in expansion cultures are Lin⁺ descendants of CD133⁺CD34⁺ cells displaying high DC differentiation capacity and should no longer be considered as multipotent CD133⁺CD34^–Lin^– cells. We further showed that the generation of CD133⁺CD34^–Lin⁺ DC precursors (DPs) was dependent on the presence of FL. The fact that CD133⁺CD34^–Lin⁺ cells possess proliferation and differentiation potential typical for DPs does support the existence of a possible DC differentiation pathway, wherein CD133⁺CD34⁺ cells give rise to CD133⁺CD34^–Lin⁺ DPs. The recently observed CD133⁺CD34^– phenotype of myeloid leukemia cells [10, 11] might represent the malignant counterpart of this DC subset rather than an aberrant CD133 expression on more mature CD34^– leukemic cells.

View larger version (30K): [in this window] [in a new window]   Figure 1. Characterization and purification of cultured CB progenitor subpopulations according to CD133 and CD34 expression. (A): Kinetics of expression of the stem cell markers CD133 and CD34 on cells generated in cultures initiated with CD133+ (day 0) and supplemented with FL, TPO, SCF, and IL-6 (FTS6) or with TPO, SCF, and IL-6 (TS6). Samples of cells from each culture condition were harvested on days 7, 14, and 21 and FACS-analyzed. Gates were set by forward and side scatter to exclude dead cells and cell aggregates. Cells were analyzed for the expression of CD133 and CD34, using dual-color flow cytometry. Limits for positivity and negativity were defined by incubating with nonimmune isotypic-matched immunoglobulin. Representative results are shown from one of 12 independent experiments. (B): Purified CD133+CD34–, CD133–CD34+, and CD133+CD34+ subpopulations from day-7 cultures. Nucleated cells collected from day-7 cultures were subjected to double immunomagnetical cell sorting for purifying CD133+CD34–, CD133+CD34+ cells generated in cultures under FTS6 condition and CD133–CD34+ cells generated in cultures under TS6 condition. Sorted cells were analyzed after labeling with anti-CD133 and anti-CD34 to evaluate their purity. In six independent experiments, mean purity for CD133+CD34–, CD133–CD34+, and CD133+CD34+ cells was 90% (86%–94%), 96% (94%–100%), and 94% (86%–96%), respectively. Results are shown from one representative experiment. Abbreviations: CB, cord blood; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL, Flt-3 ligand; IL, interleukin; PE, phycoerythrin; SCF, stem cell factor; TPO, thrombopoietin.

View larger version (46K): [in this window] [in a new window]   Figure 2. Phenotypic characterization and differentiation into myeloid dendritic precursors of purified CD133+CD34– cells generated in vitro by CD133+CD34+ cells. (A): Sorted CD133+CD34– cells from day-7 cultures in FL, TPO, SCF, and IL-6 with 87% purity after Giemsa staining were found to be monocyte-like with short cell processes. CD133+CD34– cells isolated by MACS coexpressed both CD13 and CD33 antigens. Overlay diagrams show the expression of the relevant antigen versus negative controls. (B): Day-7 purified CD133+CD34– cells differentiate into CD1a+CD11c+, CD1a–CD11c+, and CD1a+CD11c– dendritic precursors after culture with FL, GM-CSF, TNF-, and IL-4 for 14 days. Morphologically, the cells generated in the abovementioned cultures were less rounded with hyperlobulated nuclei resembling freshly isolated peripheral blood DC1 precursors. They uniformly expressed HLA-DR, CD40, CD86, CD80, and CD83 antigens while CD14 was dimly expressed on 25% of the cells. (C): CD133+CD34– cells from four different CB samples were plated in a series of limiting cell doses in cultures supplemented with FL, GM-CSF, IL-4, and TNF- for 2 weeks. DPs were identified by FACS after labeling with CD1a and CD11c. (C): A linear regression analysis was used to determine the frequency of DC progenitors. Results from four different samples were combined to generate a plot of the number of cells plated versus log percentage of negative wells with SEM (error bars). The average DP frequency is shown alongside the bars. Abbreviations: CB, cord blood; DC, dendritic cell; DP, dendritic cell precursor; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL, Flt-3 ligand; IL, interleukin; MACS, magnetic activated cell sorting; PE, phycoerythrin; SCF, stem cell factor; TNF, tumor necrosis factor; TPO, thrombopoietin.

	ACKNOWLEDGMENTS

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We thank "ELPIDA" Friends Association of Children with Cancer for their generous support to our Stem Cell Transplant program. We also thank Dr. J. Traeger-Synodinos for carefully reading the manuscript.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

	REFERENCES

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This Article Full Text (PDF) All Versions of this Article: 2005-0283v1 24/4/1137    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Goussetis, E. Articles by Graphakos, S. Search for Related Content PubMed PubMed Citation Articles by Goussetis, E. Articles by Graphakos, S.