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Identification of a Novel Subpopulation of Human Cord Blood CD34^-CD133^-CD7^-CD45⁺Lineage^- Cells Capable of Lymphoid/NK Cell Differentiation After In Vitro Exposure to IL-15 ¹

Sergio Rutella^2,*,, Giuseppina Bonanno, Maria Marone, Daniela de Ritis, Andrea Mariotti, Maria Teresa Voso^*, Giovanni Scambia, Salvatore Mancuso, Giuseppe Leone^* and Luca Pierelli^*,

^* Department of Hematology, Laboratory of Immunology, and Department of Gynecology, Catholic University Medical School, Rome, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34^- cell subsets that might reside at an earlier stage of differentiation than CD34⁺ HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34^-CD133^-CD7^-CD45^dimlineage (lin)^- HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133, 2) generation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates, 3) significant extended long-term culture-initiating cell activity, and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34⁺lin^- cells, CD34^-CD133^-CD7^-CD45^dimlin^- HSCs maintained with IL-15, but not with IL-2 or IL-7, proliferated vigorously and differentiated into a homogeneous population of CD7⁺CD45^brightCD25⁺CD44⁺ lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCR genes, IL-15-primed CD34^-CD133^-CD7^-CD45^dimlin^- HSCs failed to achieve full maturation, as manifested in their CD3^-TCR^-- phenotype. Conversely, culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively, CD34^-CD133^-CD7^-CD45^dimlin^- HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34^-lin^- HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hemopoiesis can be defined as a hierarchical process sustained by pluripotent stem cells capable of extensive self-renewal and differentiation (1). An orderly sequence of changes in gene expression occurs, leading to lineage restriction and, ultimately, production of mature blood cells. Hemopoietic stem cells (HSCs) ³ possess enormous clinical relevance, because they can reconstitute both the lymphoid and the myeloid compartment in the recipients of autologous, allogeneic, or xenogeneic transplants (1). Furthermore, HSCs can be targets for ex vivo expansion protocols and for gene therapy strategies.

CD34 Ag is a syalomucin that has been considered to be a hallmark of human transplantable HSCs (2, 3, 4); accordingly, experimental and clinical protocols have been designed exclusively for CD34⁺ subsets. Recently, several investigators have identified very primitive CD34^-lineage (lin)^- HSCs capable of long-term repopulation of nonobese diabetic/SCID mice and fetal sheep (5, 6, 7, 8, 9). The isolation of highly purified subsets of HSCs not expressing Ags associated with lineage commitment (or lin^- cells) has demonstrated a previously unrecognized heterogeneity within the human HSC compartment (10). In particular, CD133⁺CD34^-CD38^-lin^- cells isolated from human umbilical cord blood (UCB) possess in vitro growth factor requirements and progenitor capacity equivalent to the CD34⁺ stem cell fraction (10). Because CD34^- HSCs can be obscured by mature CD34^- cells, functional assays have been developed to isolate HSCs, including Hoechst 33342 dye efflux (11). Using these approaches, a rare side population of primitive CD34^- HSCs has been identified in human UCB; CD34^-lin^- side-population cells express CD7 Ag and contain lymphoid progenitors capable of rapid expansion and differentiation into functionally active NK cells, if plated on stroma supplemented with cytokines (11). In the study by Storms et al. (11), CD34^-CD7^-lin^- cells were detected at very low frequency and were excluded from analysis; thus, whether CD34^-CD7^-lin^- cells contain HSCs endowed with progenitor or stem cell activity has not been determined.

The molecular events occurring during lineage restriction involve activation or silencing of genes encoding cytokine receptors, signal transduction molecules, and transcription factors (12, 13, 14). In this respect, the isolation of progenitor populations from normal in vivo sources can greatly contribute to the understanding of the process of differentiation and lineage plasticity, by providing access to intermediates that can be characterized by functional assays and molecular approaches (15, 16, 17). In the present study, we identified a novel, rare, and primitive subset of human UCB CD34^-CD133^-CD7^-lin^- progenitors that generate T cell precursors and functionally active NK cells after culture with stromal cells supplemented with the IL-2R c-signaling cytokine IL-15. The CD34^-CD133^-CD7^-CD45^dimlin^- lymphoid progenitors that we differentiated in vitro harbored rearrangement at the TCR locus, expressed the T cell-specific transcription factor GATA-3, and could be hierarchically correlated to the recently described lymphoid-oriented CD34^-CD7⁺lin^- cells and to the transitional myeloid CD34^-CD133⁺CD7^-lin^- UCB cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
UCB processing and isolation of CD34^-CD133^-CD7^-lin^- cells

UCB samples were obtained after full-term cesarean delivery from healthy donors after written informed consent. The local human research committee approved all investigations. UCB samples were collected in sterile bags containing citrate-phosphate dextrose and adenine anticoagulant and were diluted 1/3 in PBS. Low-density mononuclear cells were separated over Ficoll-Hypaque (density, 1077 g/dl; Pharmacia, Uppsala, Sweden). Mononuclear cells were subsequently subjected to removal of lineage-committed cells by incubation with a mixture of mAb directed against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, CD41, and glycophorin A (CD235a). The manufacturer (StemCell Technologies, Vancouver, BC, Canada) provided all mAb. After incubation, a secondary Ab conjugated to a metal colloid was added, and cells were depleted of lineage marker-positive elements by a magnetic device (StemSep; StemCell Technologies). CD34⁺lin^- cells were then removed and separately collected from remaining lin^- cells by binding with PE- or FITC-conjugated anti-CD34 mAb (QBEND/10 clone; Serotec, Oxford, U.K.), followed by sorting on a FACStar^Plus flow cytometer (BD Biosciences, San Jose, CA). After extensive washings with PBS/1% BSA, isolated CD34^-lin^- cells were stained with PE-conjugated anti-CD133 mAb (Miltenyi Biotec, Bergisch-Gladbach, Germany) and FITC-conjugated anti-CD7 mAb (BD Biosciences). CD7⁺ and CD133⁺ cells were removed from the CD34^-lin^- starting population by sorting on a FACStar^Plus cytometer (BD Biosciences). The remaining cells were designated as CD34^-CD133^-CD7^-CD45^dimlin^- cells, in accordance with flow-cytometric reanalysis of surface marker expression.

Culture of purified CD34^-CD133^-CD7^-CD45^dimlin^- cells

In a preliminary set of experiments, we tested the ability of an array of human growth factors to support viability, growth, and/or differentiation of freshly isolated CD34^-CD133^-CD7^-CD45^dimlin^- cells in liquid cultures, with or without animal serum. IL-3 at 20 ng/ml, IL-6 at 20 ng/ml, IL-7 at 40 ng/ml, IL-2 at 1000 IU/ml, Flt3 ligand (Flt3-L) at 20 ng/ml, platelet-derived growth factor at 3 ng/ml, epidermal growth factor at 0.4 ng/ml, thrombopoietin (TPO) at 10 ng/ml, GM-CSF at 20 ng/ml, stem cell factor (SCF) at 10 ng/ml, or IL-15 at 50 ng/ml (all purchased from R&D Systems, Oxon, Cambridge, U.K.) were tested individually in 72-h cultures for their capacity to sustain viability and growth (as revealed by cell counts, exclusion of propidium iodide, and FACS analysis of light scatter characteristics) or to promote differentiation, which was monitored through the detection of CD34, CD133, and CD7 expression, as will be detailed. Cytokine concentrations were selected based on previously published studies (18). Cultures were performed in -MEM culture medium supplemented with 10% horse serum, 10% bovine serum, and 10^-6 M hydrocortisone, in the presence or absence of the indicated cytokine combinations (19). When the adequate culture conditions were established and standardized based on preliminary experiments, culture length was prolonged to 7 days, using the optimal growth factor supplementation.

CFU assays, long-term culture-initiating cell assays, and lymphoid cell cultures

Colony-forming cells (CFU-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E)) were generated from purified UCB cells (CD34^-CD133^-CD7^-CD45^dimlin^- and CD34⁺lin^- cells; the latter used as reference control) before and after cytokine treatments by 14-day methylcellulose semisolid cultures in the presence of 25% serum substitute to avoid interference from serum factors (Bit 9500; StemCell Technologies) and SCF, IL-3, GM-CSF, G-CSF, Flt3-L, and erythropoietin, as reported (18, 20). Replicates of cloning assays were established under the same culture conditions but in the presence of SCF, TPO, and IL-6 to identify megakaryocytic aggregates (Meg-aggregates), as previously described (18). Cloning activity was expressed as cumulative colony efficiency (CFU-GM plus BFU-E plus Meg-aggregates) both in terms of absolute number of colonies per number of seeded cells and in terms of percentage of seeded cells. Extended (12-wk) long-term culture-initiating cell (ELTC-IC) activity was assessed by limiting-dilution analysis (LDA) and 12-wk long-term culture on a murine stromal cell line engineered to produce human G-CSF and IL-3 (M2-10B4 cell line; a gift from Dr. C. Eaves (Terry Fox Laboratory, Vancouver, BC, Canada)) (18, 21). At wk 6, cultures were trypsinized, and cells were reseeded on a new irradiated layer of M2-10B4 cells to circumvent cell senescence. Freshly isolated CD34^-CD133^-CD7^-CD45^dimlin^- cells were tested in NK culture assays, which consisted of 21-day stroma-based culture on MS-5 murine cells in -MEM supplemented with 10% horse serum, in the presence or absence of IL-15 at 50 ng/ml. The starting population of CD34^-CD133^-CD7^-CD45^dimlin^- cells was seeded at 1 x 10⁵ per well in 24-well plates, where a confluent stromal layer of MS-5 cells had been grown previously.

Limiting-dilution assays

Suspension cultures containing IL-15 were set up with purified CD34^-CD133^-CD7^-CD45^dimlin^- cells in 20 µl of medium in the individual wells of a 60-well Terasaki microwell plate. After 7 days of culture, each well was examined for the presence of viable cells and then assayed individually for the presence of lymphoid progenitors. Wells were scored as positive for IL-15 responsiveness when >20 viable cells could be identified on day 7 and when cells expressed detectable levels of the lymphoid-associated Ag CD7 by flow cytometry. All other wells were scored as negative and usually contained no detectable cells. The frequency of IL-15-responsive cells in the CD34^-CD133^-CD7^-CD45^dimlin^- cells was calculated from the proportion of negative wells based on Poisson statistics, as previously reported (18).

Immunological markers

Aliquots of cells were incubated for 30 min at 4°C with pretitrated saturating dilutions of the following FITC-, PE-, PerCP-, or PE-Cy5 (Tricolor)-conjugated mAb: CD16 (NKP15 clone, IgG1), CD19 (4G7 clone, IgG1), CD22 (S-HCL-1 clone, IgG2b), CD34 (8G12 clone, IgG1), CD38 (HB7 clone, IgG1), CD56 (My31 clone, IgG1), CD45RA (L48 clone, IgG1), CD45RO (UCHL-1 clone, IgG1), CD25 (2A3 clone, IgG1), CD44 (L178 clone, IgG1), glycophorin-A (CD235a; GAR-2 clone, IgG2b), CD132 (IL-2R -chain; AG184 clone, IgG1), TCR (WT31 clone, IgG1), TCR (11F2 clone, IgG1; BD Biosciences), CD94 (HP-3D9 clone, IgG1), CD158a (HP-3E4 clone, IgM), CD158b (CH-L clone, IgG2b; BD PharMingen, San Diego, CA), CD3 (S4.1 clone, IgG1), CD4 (S3.5 clone, IgG2a), CD8 (3B5 clone, IgG2a), CD7 (CD7-6B7 clone, IgG2a), CD90 (F15-42-1 clone, IgG1), CD64 (10.1 clone, IgG1), CD65 (VIM2 clone, IgG1), HLA-DR (TU36 clone, IgG2b), CD11b (CR3 Bear-1 clone, IgG1), CD13 (TUK1 clone, IgG1), CD45 (HI30 clone, IgG1), Sca-1 (Ly-6A.2 clone, IgG2a; Caltag Laboratories, Burlingame, CA), CD105 (SN6 clone, IgG1; Serotec), and CD133 (AC133 clone, IgG1; Miltenyi Biotec). Appropriate fluorochrome-conjugated isotype-matched mAb purchased from the different manufacturers were used as control for background staining. After extensive washings with PBS/1% BSA, cells were kept on ice until flow-cytometric analysis.

Assay for NK cell function

NK cell function was evaluated by flow cytometry, following a previously described protocol, which measures target cell death through the uptake of membrane-impermeable dyes (10). Briefly, 1 x 10⁶ NK-sensitive K562 cells were used as target cells and were labeled with 2.5 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 10 min at room temperature. For lysis assay, K562 cells were plated in duplicate in RPMI 1640 culture medium supplemented with Bit H9500 serum substitute at the various E:T ratios described in the figures. The cocultures were then incubated for 24 h at 37°C with 5% CO₂. Cells were pelleted, washed with PBS supplemented with 1% BSA, and further incubated with 20 µg/ml 7-amino actinomycin-D (7-AAD; Molecular Probes) for an additional 30 min, before flow cytometry analysis.

Measurement of lymphocyte divisions by CFSE

For cell-doubling experiments, cells were resuspended in PBS containing 2.5 µM CFSE (Molecular Probes) for 10 min at room temperature (22). To quench the labeling process, an equal volume of FCS was added; after washings with PBS supplemented with 3% FCS, cells were incubated overnight with medium alone and then exposed to the various cytokine combinations, as detailed in the figures.

Semiquantitative RT-PCR

mRNA expression levels were evaluated by RT-PCR, normalizing the levels of test RNA to that of the internal control, aldolase A. Total RNA extractions were conducted with the RNeasy mini-kit (Qiagen, Hilden, Germany), and RNA obtained from 2 x 10⁴ cells was reverse transcribed with 25 U of Moloney murine leukemia virus reverse transcriptase (PE Applied Biosystems, Foster City, CA) at 42°C for 30 min in the presence of random hexamers. Two microliters of these cDNA products were amplified with 1 U of AmpliTaq Gold (PE Applied Biosystems) in the presence of the specific primers for the mRNA of interest, together with the aldolase-A primer (23). Reactions were conducted in the GeneAmp PCR System 9600 (PE Applied Biosystems). Conditions were chosen so that none of the amplification products obtained from the RNAs of interest reached a plateau, and that the two primer sets did not compete with each other. Amplification of GATA-3 mRNA was achieved by 30 cycles of 45 s at 95°C, 45 s at 55°C, and 1 min at 72°C in the presence of 3 mM MgCl₂. Amplification conditions and primer sequences for the other markers studied in the present report were described elsewhere (24, 25, 26, 27, 28). The PCR products were loaded onto 1.5% agarose gels and stained with ethidium bromide. Each set of reactions included a no-sample negative control. The ratio between the sample RNA to be determined and aldolase-A was calculated to normalize for initial variations in sample concentration and as a control for reaction efficiency. The following primers were used for amplification of GATA-3 mRNA: 5'-GTCCTGTGCGAACTGTCAGA-3' and 5'-TAAACGAGCTGTTCTTGGGG-3'. All oligonucleotide primers were synthesized by M-Medical (Florence, Italy).

DNA extraction and amplification

DNA was extracted using DNAzol (Life Technologies, Eggenstein, Germany), following the manufacturers’ instructions. TCR rearrangement was studied using a nested PCR technique (29). Briefly, DNA was amplified using the following primers: 5'-AGGGTTGTGTTGGAATCAGG-3' (TCR-11) and 5'-CGTCGACAACAAGTGTTGTTCCAC-3' for the primary PCR, and the TCR-11 primer and 5'-GGATCCACTGCCAAAGAGTTTCTT-3' for the nested PCR. For both amplifications, PCR consisted of 30 cycles at 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min and resulted in a fragment ranging between 150 and 180 bp following the nested PCR. As control, PCR of the housekeeping gene Bcl-2 was performed, using 5'-GCAATTCCGCATTTAATTCATGG-3' and 5'-GAAACAGGCCACGTAAAGCAAC-3' as primers. PCR was performed for 35 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min. This resulted in a fragment of 154 bp. PCRs were conducted in a 25-µl mixture containing 200 ng of genomic DNA, and a master mix of PCR buffer, 1.5 mM MgCl₂, 250 nM dNTPs, 1.25 U of Taq polymerase (Taq Platinum; Life Technologies), and 0.8 mM each oligonucleotide. A positive control and a negative control, containing water instead of DNA, were included in all PCR. PCR products were analyzed on a 3% agarose gel, stained with ethidium bromide.

Flow cytometry and immunofluorescence analysis

Cells were run through a FACScan flow cytometer (BD Biosciences), equipped with an argon laser emitting at 488 nm. Details on instrument requirements and settings were published elsewhere (30). FITC, PE, and PerCP, or PE-Cy5 (Tricolor) signals were collected at 530, 575, and 670 nm, respectively; spectral overlap was minimized by electronic compensation with Calibrite beads (BD Biosciences) before each determination series. A minimum of 10,000 events was collected and acquired in list mode using the CellQuest software (BD Biosciences).

Statistical methods

The approximation of population distribution to normality was tested preliminarily using statistics for kurtosis and symmetry. Data were presented as mean ± SD. Consequently, all comparisons were performed with Student’s t test for paired or unpaired data or with the ANOVA, as appropriate. The criterion for statistical significance was defined as p 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of CD34^-CD133^-CD7^-lin^- cells

UCB mononuclear cells were enriched in CD34^- cells and lin^- cells by magnetic depletion of cells labeled with a mixture of mAb directed against lymphoid, myeloid, and erythroid Ags, and by flow-cytometric removal of cells reactive with anti-CD34 mAb. The purity of sorted CD34^-lin^- cells always exceeded 98%, as assessed by FACS analysis (data not shown). CD133 and CD7 were found on 1.6–22.3% and on 8.5–39% of CD34^-lin^- cells, respectively, in the different UCB samples tested. Furthermore, freshly isolated CD34^-lin^- cells were reactive, albeit at low frequency, with the anti-CD44 (11 ± 5%) and anti-CD25 mAb (5 ± 2%). Next, we depleted CD34^-lin^- cells from CD7⁺ and CD133⁺ cells using a flow cytometry-based cell-sorting strategy. Gating windows for cell-sorting experiments were constructed as depicted in Fig. 1A. Coselection for absence of CD7 and CD133 resulted in a highly purified CD34^-CD133^-CD7^-lin^- cell population (Fig. 1B). Contamination by residual CD7-expressing lymphoid progenitor cells could be excluded based on FACS reanalysis (Fig. 1B) and on PCR analysis of TCR rearrangement status in CD34^-CD133^-CD7^-lin^- cells (Fig. 1C). CD34^-CD133^-CD7^-lin^- cells comprised 0.16% on average (range, 0.03–0.28%) of UCB mononuclear cells collected after full-term deliveries from 10 normal donors.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Isolation and characterization of CD34-CD133-CD7-lin- cells. UCB CD34-lin- cells were depleted from CD7+ and CD133+ cells through cell sorting on a FACStarPlus flow cytometer (BD Biosciences). A, Sorting windows (R1) were established to include >95% of the CD7-CD133- events. B and C, CD34-CD133-CD7-lin- cells were virtually devoid of contaminating residual T cells, as shown by FACS reanalysis and by molecular studies on TCR rearrangement status. Lane 1, Purified CD34+ cells; lane 2, CD34-CD133-CD7+lin- cells; lane 3, CD34-CD133-CD7-lin- cells; lane 4, Jurkat cells; lane 5, monoclonal control; lane 6, polyclonal control; and lane 7, dH2O. As control, PCR of the housekeeping gene Bcl-2 was performed and is shown in C (bottom).

The expression of informative differentiation Ags on highly purified CD34^-CD133^-CD7^-lin^- UCB cells was investigated by multiparameter flow cytometry. The T cell and B cell lineage-associated Ags CD3 and CD22 as well as CD235a and CD11b, assigned to erythroid and granulocytic cells, respectively, were undetectable on CD34^-CD133^-CD7^-lin^- UCB cells (Fig. 2). Accordingly, TCR and TCR as well as CD4 and CD8 Ags were absent. A readily discernible subset of CD34^-CD133^-CD7^-lin^- UCB cells was reactive with anti-CD105 (10 ± 3%) and anti-CD90 mAb (12 ± 4%); similarly, expression of HLA-DR and Sca-1 was shown on a small subset of cells (<10%), in accordance with previous reports on putative stem/progenitor cells capable of regenerating human myocardium (31). Of interest, CD34^-CD133^-CD7^-lin^- UCB cells uniformly expressed low levels of CD45 (mean fluorescence intensity equal to 27 ± 6 compared with 130 ± 15 in peripheral blood CD3⁺ T cells used as a control) and were virtually negative for Ags assigned to the granulocytic/monocytic differentiation pathway, e.g., CD33, CD13, CD64, CD65, as well as for the NK cell-associated Ags CD16 and CD56 (Fig. 2).

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Phenotypic features of freshly isolated CD34-CD133-CD7-lin- cells. CD34-CD133-CD7-lin- cells were further analyzed for the expression levels of an array of differentiation/maturation Ags. Cells were gated in R1 based on forward-scatter (FSC)/side-scatter (SSC) characteristics. One representative experiment of six with similar results is shown. Markers were set according to the proper isotypic control. The percentage of cells staining positively for a given Ag is indicated in each bidimensional cytogram.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Molecular profile of freshly isolated CD34-CD133-CD7-lin- cells. mRNA levels for survival genes, cell cycle modulators, transcription factors, and differentiation proteins were measured with semiquantitative RT-PCR. The TF-1 leukemic cell line and CD34+lin- cells were used as reference control for gene expression levels. Absorbance relative to the housekeeping gene (aldolase A) was plotted in A. Data represent mean ± SD from 10 independent experiments performed in duplicate. One experiment of 10 with similar results is shown in B. Lane 1, CD34+ cells; lane 2, CD34-CD133-CD7-CD45dimlin- cells; and lane 3, TF-1 cell line.

Next, we tested numerous growth factors for the ability to support survival and/or differentiation of human CD34^-CD133^-CD7^-CD45^dimlin^- cells in liquid cultures performed in the presence of horse/bovine serum and 10^-6 M hydrocortisone (19). Based on data from preliminary experiments (not shown), corticosteroids and animal serum were included in all growth experiments, because their concomitant presence during the 48-h culture period reduced spontaneous cell death by 20% on average in any experimental condition tested as compared with medium supplementation with serum substitute alone. When used alone or in combinations, IL-3, IL-6, TPO, Flt3-L, IL-7, GM-CSF, IL-2, platelet-derived growth factor, and epidermal growth factor exerted no appreciable effects on growth, viability, and differentiation of CD34^-CD133^-CD7^-CD45^dimlin^- cells following 72 h of culture, as measured by viable cell counts and CD133 and CD7 expression. Of interest, SCF preserved cell viability and promoted the appearance of a population of cells that averaged 12% and expressed low levels of CD133. At variance with the TCR chain-signaling cytokines IL-2 and IL-7, IL-15 was capable of promoting CD34^-CD133^-CD7^-CD45^dimlin^- cell growth, with an average 2.4-fold increase in cell number, maintained cell viability, and induced expression of the T cell-associated Ag CD7 on an average of 15% of cultured cells on day 3.

Eight individual samples of purified CD34^-CD133^-CD7^-CD45^dimlin^- cells isolated from eight different UCB donors were assayed for direct cloning efficiency in semisolid methylcellulose culture in the presence of a combination of cytokines, as already detailed. Following 14 days of serum-free culture, no colonies of any lineage were scored when CD34^-CD133^-CD7^-CD45^dimlin^- cells were seeded at the concentration of either 1,000 or 5,000 cells per plate (Table I). Conversely, after increasing cells to 10,000 per plate, five small nonhemoglobinized colonies on average were identified in replicate plates containing SCF, erythropoietin plus G-CSF, GM-CSF, IL-3, and Flt3-L (cloning efficiency, 0.05%). On the contrary, the CD34⁺lin^- counterpart isolated from the same UCB samples and used as a control cell population produced confluent colonies when seeded at 5,000 and 10,000 cells per plate, and hemoglobinized and nonhemoglobinized colonies averaged 100 at a starting concentration of 1,000 cells per plate (cloning efficiency, 10%; Table I). To assay for long-term functional activity of CD34^-CD133^-CD7^-CD45^dimlin^- cells, we performed ELTC-IC assays through LDA and 12-wk culture on the murine stromal cell line M2-10B4, engineered to produce human G-CSF and IL-3. From these assays, we learned that human CD34^-CD133^-CD7^-CD45^dimlin^- cells were endowed with low but measurable long-term activity (average ELTC-IC frequency of 1:12,000 in five independent experiments). For purposes of comparison, the CD34⁺lin^- counterpart from the same UCB samples showed an average frequency of 1:600 ELTC-IC, in accordance with previously published reports (18, 24).

Based on the results obtained with SCF and IL-15 after 72-h liquid culture, we first performed a more in-depth analysis of the effects produced by SCF on survival and expansion of CD34^-CD133^-CD7^-CD45^dimlin^- cells maintained in culture for up to 7 days. This experimental condition was established in three replicate experiments with UCB cells from three different donors. In the absence of exogenous cytokines, cell count declined by 40% on average as compared with the starting cell population, and cell viability averaged 40% (Table II). Conversely, cultures maintained in the presence of SCF showed significant increases of cell counts, associated with cell viability scores that were consistently >80%. The onset of cell proliferation after exposure to SCF was also confirmed by cell-doubling experiments with the fluorescent dye CFSE. Whereas cells kept in the absence of SCF preserved their CD34^-CD133^- phenotype throughout the culture period, cells maintained with SCF acquired the CD133 marker by day 4 in the absence of significant CD34 expression. Notably, the majority of SCF-treated cells coexpressed both CD133 and CD34 by day 7 (Table II; Fig. 4).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Phenotypic features of SCF-treated CD34-CD133-CD7-CD45dimlin- cells. The expression of the stem cell-related Ag CD34 and CD133 was evaluated by flow cytometry after culturing for 7 days in the presence (left panels) or in the absence (right panels) of SCF at 10 ng/ml. All cultures were performed in -MEM culture medium supplemented with 10% horse serum, 10% bovine serum, and 10-6 M hydrocortisone (19 ). The percentage of cells reactive with the anti-CD34 and/or the anti-CD133 mAb at the different time points is indicated in each bidimensional cytogram. Markers for quadrant analysis were set according to the proper isotypic controls. FSC, Forward scatter; SSC, side scatter. One representative experiment of four with similar results is shown.

Molecular analyses confirmed the commitment of SCF-treated CD34^-CD133^-CD7^-CD45^dimlin^- cells toward the myeloid lineage, as indicated by high levels of transcripts for myeloperoxidase, when compared with cells cultured in the absence of exogenous cytokines or with TF-1 erythroleukemia cells, used as a control cell population (Fig. 5). Also, reactivation of Bcl-2 gene transcription, lack of mRNA signals for TGF-1, and significant down-regulation of p21^Cip1 and p27^Kip1 mRNA levels occurred in SCF-treated CD34^-CD133^-CD7^-CD45^dimlin^- cells, compared with cells maintained without SCF (Fig. 5) and with the starting cell population (Fig. 3). Conversely, no changes were observed in GATA-3 mRNA levels. As anticipated by the FACS analysis of CD34 Ag expression, mRNA signals for CD34 were consistently detected after priming with SCF, indicating active gene transcription (Fig. 5).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Molecular profile of SCF-treated CD34-CD133-CD7-CD45dimlin- cells. mRNA levels for survival genes, cell cycle modulators, transcription factors, and differentiation proteins were measured with semiquantitative RT-PCR after 7 days of culture with SCF at 10 ng/ml. The TF-1 cell line was used as reference control for gene expression levels. Data on cells cultured without exogenous cytokines were presented for purposes of comparison. Absorbance relative to the housekeeping gene (aldolase A) was plotted in A. Data represent mean ± SD from four independent experiments performed in duplicate. One representative experiment is depicted in B. Lane 1, CD34-CD133-CD7-CD45dimlin- cells (no cytokine); lane 2, SCF-treated CD34-CD133-CD7-CD45dimlin- cells; and lane 3, TF-1 cell line.

Following the observation that IL-15 promoted cell survival and induced CD7 expression in liquid culture, CD34^-CD133^-CD7^-CD45^dimlin^- cells were maintained for 7 days in the presence of exogenous IL-15 at 50 ng/ml. Under these experimental conditions, a population of putative lymphoid progenitors emerged that homogeneously expressed the T cell-associated Ag CD7, together with markers conventionally assigned to naive T cells, e.g., CD45RA (Table III). Accordingly, minute fractions of cells expressed the memory T cell-associated Ag CD45RO. Interestingly enough, high percentages of cells differentiated with IL-15 stained positively with both the anti-CD25 and the anti-CD44 mAb. These features were reminiscent of the early stages of normal T cell thymic development (12). In line with the exquisite sensitivity to IL-15, CD34^-CD133^-CD7^-CD45^dimlin^- cells expressed high levels of the IL-2/IL-15R chain (Table III). However, cells completely lacked expression of CD3, as well as of CD4 and CD8 Ag; consistently, either surface or chains of the TCR complex were undetectable. CD45 expression levels were significantly increased after culture, and IL-15-primed cells homogeneously acquired a CD45^bright staining pattern. Conversely, most CD34^-CD133^-CD7^-CD45^dimlin^- cells cultured without IL-15 retained a CD45^dim phenotype (Table III). As shown in Table III, spontaneous cell differentiation occurred and cells maintained in the absence of exogenous IL-15 up-regulated the expression of the lymphoid-cell associated Ag CD7. Furthermore, 24 ± 5% of CD34^-CD133^-CD7^-CD45^dimlin^- cells cultured with medium alone displayed a CD44⁺CD25⁺ phenotype as compared with 5 ± 2% of freshly isolated CD34^-CD133^-CD7^-CD45^dimlin^- cells. Data on phenotype and viability of IL-15-primed CD34^-CD133^-CD7^-CD45^dimlin^- cells are summarized in Table III.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. TCR chain rearrangement after priming with IL-15. TCR rearrangement status was investigated using a nested PCR technique, as already reported (29 ). A positive control and a negative control, containing water instead of DNA, were included in all PCR. PCR products were analyzed on a 3% agarose gel, stained with ethidium bromide. CD34-CD133-CD7+lin- cells (lane 2) represented bona fide committed lymphoid precursors and were used as positive controls. Lane 1, MW marker; lane 2, CD34-CD133-CD7+lin- cells; lane 3, IL-15-primed CD34-CD133-CD7-lin- cells; lane 4, monoclonal control; lane 5, polyclonal control; and lane 6, dH2O.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Molecular profile of IL-15-treated CD34-CD133-CD7-CD45dimlin- cells. mRNA levels for survival genes, cell cycle modulators, transcription factors, and differentiation proteins were measured with semiquantitative RT-PCR. The TF-1 cell line was used as reference control for gene expression levels. Data on cells cultured without IL-15 were presented for purposes of comparison. Absorbance relative to the housekeeping gene (aldolase A) was plotted in A. Data represent mean ± SD from four independent experiments performed in duplicate. One representative experiment is shown in B. Lane 1, CD34-CD133-CD7-CD45dimlin- cells (no cytokine); lane 2, IL-15-treated CD34-CD133-CD7-CD45dimlin- cells; and lane 3, TF-1 cell line.

LDAs performed with CD34^-CD133^-CD7^-CD45^dimlin^- cells plated at the concentration of one cell per well in the presence of IL-15 supplementation indicated that the average frequency of IL-15-responsive CD34^-CD133^-CD7^-CD45^dimlin^- cells was 1:8 (range, 1:6–1:11) in three independent experiments performed in triplicate with three individual UCB samples. Finally, we wanted to clarify whether CD105⁺ mesenchymal stem cells contained within the starting population of CD34^-CD133^-CD7^-CD45^dimlin^- cells (Fig. 2) possessed the ability to sustain the lymphoid differentiation process of CD34^-CD133^-CD7^-CD45^dimlin^- cells by providing soluble factors or accessory signals. To accomplish this goal, we depleted CD105⁺ cells from CD34^-CD133^-CD7^-CD45^dimlin^- cells before culturing in the presence of IL-15 (Fig. 8A). These experiments indicated that CD105^-CD34^-CD133^-CD7^-CD45^dimlin^- cells maintained the ability to differentiate along the lymphoid lineage, as shown by immunophenotypic (Fig. 8B) and molecular studies (not shown).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Lymphoid differentiation potential of CD34-CD133-CD7-CD45dimlin- after depletion of CD105+ cells. CD34-CD133-CD7-CD45dimlin- cells were initially depleted of contaminating CD105+ cells and then cultured with exogenous IL-15 as already described. Cells were virtually devoid of contaminating CD105+ as well as CD7+ cells, as shown by FACS reanalysis (A). The expression of the lymphoid-associated Ag CD7 on CD105-CD34-CD133-CD7-CD45dimlin- cells cultured with IL-15 was evaluated by multiparameter flow cytometry; results from one representative experiment of three with similar results is shown in B.

To detect whether CD34^-CD133^-CD7^-CD45^dimlin^- cells could give origin to terminally differentiated NK cells, a stroma-based 21-day culture system was established in the presence or absence of IL-15. On day 21, the expression of informative NK cell-associated Ags was investigated on cells recovered from the stromal cell support. CD94 Ag was detected on 30% of lymphoid progenitors on average, whereas the human neural-cell adhesion molecule CD56 was expressed at high levels by the majority of cells exposed to IL-15 (Fig. 9A). In particular, >90% CD56-expressing cells resided in the CD56^bright fraction, whereas CD16, the low-affinity FcRIII, could be detected on 10% of cells on average (Fig. 9A). In addition, the NK cell-associated killer Ig-like receptors CD158a and CD158b were expressed on 10% of cells (data not shown). Collectively, these phenotypic features along with the large granular lymphocyte morphology (data not shown) were reminiscent of NK cells. In contrast, CD34⁺lin^- cells were incapable of giving rise to NK cells when cultured on stroma cells supplemented with IL-15, as shown in Fig. 9B. In addition, stromal cells not supplemented with IL-15 induced no measurable NK cell differentiation from the CD34^-CD133^-CD7^-CD45^dimlin^- cells, as shown by FACS analysis of NK cell-associated surface Ags (Fig. 9C). CD34^-CD133^-CD7^-CD45^dimlin^- cells cultured on stroma supplemented with a combination of IL-2 (1000 IU/ml) and IL-7 (40 ng/ml) developed no phenotypic features indicative of NK cell differentiation, further suggesting the specificity of IL-15 in the determinism of the reported phenomena (Fig. 9D).

View larger version (48K): [in this window] [in a new window]   FIGURE 9. Phenotypic features of CD34-CD133-CD7-CD45dimlin- cells cultured on a stromal cell support supplemented with IL-15. The expression of informative differentiation Ags on CD34-CD133-CD7-CD45dimlin- cells was evaluated by multiparameter flow cytometry after culturing for 21 days on stromal cells in the presence (A) or absence (B) of IL-15 at 50 ng/ml. Parallel cultures were established with CD34-CD133-CD7-CD45dimlin- cells in the presence of IL-2 at 1000 IU/ml and IL-7 at 40 ng/ml (C). As a control, CD34+lin- cells cultured in the presence of IL-15 and stromal cell support (D) showed only modest induction of NK cell Ags. Markers for quadrant analysis were set according to the proper isotypic controls (not shown). The percentage of cells expressing a given Ag is indicated in each bidimensional cytogram. One representative experiment of four with similar results is shown.

View larger version (24K): [in this window] [in a new window]   FIGURE 10. Cytotoxicity of NK progenitors against K562 target cells. K562 cells were loaded with CFSE, as detailed in Materials and Methods. CD34-CD133-CD7-CD45dimlin- cells primed with IL-15 (putative NK progenitor cells) and stromal cells were cocultured with graded numbers of NK-sensitive K562 cells or with NK-resistant (NK-res) Raji cells for 24 h, as indicated. A, The percentage of specific lysis was calculated by subtracting the fraction of 7-AAD+CFSE+ cells in control cultures not containing NK progenitors from the fraction of 7-AAD+CFSE+ cells in the cocultures containing NK progenitors (mean ± SD recorded in four independent experiments performed in duplicate). *, p < 0.05 compared with cultures performed in the absence of IL-15-primed progenitor cells and with cultures containing IL-15 primed HSCs and K562 cells at a 1:1000 E:T ratio. B, One representative experiment of four with similar results is shown. The percentage of lysed K562 cells (7-AAD+CFSE+ cells) is indicated in the upper right quadrant of each bidimensional cytogram.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The objectives of the present investigation were the following: 1) to identify a relationship between the novel CD34^-CD133^-CD7^-CD45^dimlin^- HSC subset and the previously described CD34^-CD133⁺CD7^-lin^- and CD34^-CD7⁺lin^- cells and 2) to evaluate the instructive action of individual cytokines on phenotype, function, and molecular profile of human CD34^-CD133^-CD7^-CD45^dimlin^- HSCs. To this purpose, suitable methodological approaches to investigate T cell differentiation potential were cytokine-sustained, serum-containing liquid cultures and the cultivation on established stromal cell layers in adjunct to cytokine supplementation, as previously reported by Storms et al. (11) for the assessment of the differentiation repertoire of CD34^-CD7⁺lin^- UCB cells.

In this study, we isolated and characterized a previously undescribed subset of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs from human UCB. These cells significantly differed from those studied by other investigators (10, 11), because they were selected based on the lack of CD133 and CD7 expression. At variance with the CD34^-CD7⁺ lymphoid progenitors described elsewhere (11), CD34^-CD133^-CD7^-CD45^dimlin^- HSCs did not express measurable amounts of CD11b, but rather they displayed a CD105^lowCD90^lowCD117^low phenotype.

Preliminarily, CD34^-CD133^-CD7^-CD45^dimlin^- HSCs were plated in suspension cultures with the objective to determine growth factor requirements. Among the various cytokines that we used, SCF and IL-15 were the only growth factors capable of sustaining cell viability. In line with these findings, both IL-2/IL-15R chain (CD132) and SCFR (c-kit or CD117) were found on freshly isolated CD34^-CD133^-CD7^-CD45^dimlin^- cells. These data prompted us to evaluate the effects of SCF and IL-15 on the developmental fate of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs, as prototypical myeloid-supporting and lymphoid-supporting cytokines, respectively. When cultured in the presence of SCF, CD34^-CD133^-CD7^-CD45^dimlin^- cells acquired a CD34⁺CD133⁺ phenotype, generated CFU-GM, BFU-E, and Meg-aggregates, manifested ELTC-IC activity, and up-regulated mRNA levels for myeloperoxidase. Furthermore, SCF-primed HSCs down-regulated mRNA signals for cell-cycle inhibitors, up-regulated Bcl-2 levels, and proliferated, as evaluated through cell-doubling experiments with CFSE. Particularly, differentiation of CD34^-CD133^-CD7^-CD45^dimlin^- cells toward the myeloid lineage occurred with sequential and coordinated appearance of CD133 and CD34 Ags. This interpretation of the data implies that CD34^-CD133^-CD7^-CD45^dimlin^- cells represent a more primitive HSC population than the previously described CD34^-CD133⁺CD7^-lin^- HSCs.

Interestingly, IL-15 promoted growth and differentiation of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs into lymphoid progenitors with TCR rearrangement and expression of CD45RA and the T cell-associated Ag CD7. Of interest, these cells lacked markers typically found on mature lymphoid cells, e.g., surface membrane/intracellular TCR chains, CD3, CD4, or CD8 Ags, suggesting absence of terminal T cell differentiation. mRNA signals for the T cell-associated transcription factor GATA-3, one of the crucial molecules implicated in the early phases of T lymphopoiesis (38, 39), as well as gene activity for the survival/antiapoptotic factors TGF-1 and Bcl-2 were strongly up-regulated after priming with IL-15. Similar to our findings, high GATA-3 mRNA levels were recently detected in common lymphoid progenitors differentiated from UCB CD34⁺CD38^-CD7⁺ HSCs (40). CD34^-CD133^-CD7^-CD45^dimlin^- cells primed with IL-15 acquired a CD44⁺CD25⁺ phenotype that closely resembled triple-negative thymic progenitors (41). Also, CD34^-CD133^-CD7^-CD45^dimlin^- HSCs displayed a modest ability to differentiate into cells with a CD44⁺CD25⁺CD7⁺ phenotype in cultures maintained in the absence of exogenous IL-15, although cell viability was reproducibly poor. These observations imply that the effects of IL-15 on lymphoid differentiation of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs might be, at least in part, accounted for by the promotion of cell survival.

Experiments documenting lack of terminal T cell maturation after priming with IL-15 alone prompted us to culture CD34^-CD133^-CD7^-CD45^dimlin^- cells on a stromal support in the presence or the absence of IL-15 supplementation. Consistent with the known actions of IL-15 on NK cell development and function (42), CD34^-CD133^-CD7^-CD45^dimlin^- cells cultured with stromal cells and IL-15 differentiated into a homogeneous population of CD3^-CD56⁺CD16^+/-CD94^+/- cells, reminiscent of immature NK cells (43, 44). Killer Ig-like receptor acquisition was polyclonal, because cells coexpressed different receptors, e.g., CD158a and CD158b (data not shown). In our system model, stromal support was not dispensable for IL-15 to promote NK cell differentiation, because no phenotypic features of NK cells were detected after priming of CD34^-CD133^-CD7^-CD45^dimlin^- cells with IL-15 in the absence of stromal cells. Other cytokines belonging to the family of IL-2/IL-15R chain-binding cytokines, e.g., IL-2 and IL-7, were ineffective at promoting T/NK cell differentiation, when used either alone or in combination, as also found by others (45). In contrast, stromal cells were incapable of sustaining NK cell development in the absence of IL-15 supplementation. Notably, the CD34⁺lin^- counterpart exhibited minimal NK cell differentiation potential in the presence of a stromal layer and IL-15, when compared with CD34^-CD133^-CD7^-CD45^dimlin^- cells. When tested for their ability to lyse sensitive leukemic targets, cells differentiated with IL-15 and stromal cells exhibited measurable lytic responses, even at low E:T ratio, indicating the acquisition of the functional features of NK cells.

It must be emphasized that the failure to detect CD7⁺ lymphoid progenitors by flow cytometry within the starting population of CD34^-CD133^-CD7^-CD45^dimlin^- HSCs as well as the absence of mRNA signals for the TCR and the high frequency of IL-15-responsive progenitor cells measured with LDA argue against the possibility that the functional properties here assigned to CD34^-CD133^-CD7^-CD45^dimlin^- HSCs reflect the cytokine-driven expansion of contaminating T cells.

Collectively, CD34^-CD133^-CD7^-CD45^dimlin^- UCB cells were superimposable in terms of differentiation potential to the putative T cell/NK cell common progenitor that has been recently cloned from fetal liver and from mouse fetal thymus (46, 47). Conceivably, UCB CD34^-CD133^-CD7^-CD45^dimlin^- cells represent a primitive HSC population with an inherent NK/T cell differentiation capability. Coupled with previous descriptions of the coexistence of lymphoid-oriented CD34^-CD7⁺lin^- cells and myeloid-oriented CD34^-CD133⁺CD7^-lin^- HSCs in UCB, our findings support a hierarchical view of the stem cell compartment, in which the very primitive CD34^-CD133^-CD7^-CD45^dimlin^- cells that we characterized in this study for the first time might reside in dynamic equilibrium with lymphoid-restricted (CD34^-CD7⁺lin^-), transitional myeloid (CD34^-CD133⁺CD7^-lin^-), and myeloid-oriented (CD34⁺CD133⁺) stem cells. Studies aimed at evaluating whether CD34^-CD133^-CD7^-CD45^dimlin^- cells possess multilineage in vivo reconstitution potential are underway in animal transplantation models.

A final implication of our findings pertains to the transplantation of highly purified CD34⁺ HSCs, which has enormously expanded in recent years for the treatment of malignant and nonmalignant disease (48). Based on the data presented in this study, it can be assumed that depletion of CD34^- HSC subsets with lymphoid differentiation potential contained within the unmanipulated HSC graft is expected to contribute to the delayed immunological reconstitution that is routinely observed after CD34-selected HSC transplantation (48). It remains to be evaluated whether ex vivo cytokine-driven expansion of CD34^- HSC subsets endowed with lymphoid developmental potential might be exploited to accelerate lymphoid recovery and boost antitumor responses after HSC transplantation.

	Footnotes

 
¹ This work was supported by Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Italy) and by the Stem Cell Project (Fondazione Cassa di Risparmio di Roma, Rome, Italy).

² Address correspondence and reprint requests to Dr. Sergio Rutella, Department of Hematology, Laboratory of Immunology, Catholic University Medical School, Largo A. Gemelli 8, 00168 Rome, Italy. E-mail addresses: sergiorutella@tin.it or srutella{at}rm.unicatt.it

³ Abbreviations used in this paper: HSC, hemopoietic stem cell; lin, lineage; UCB, umbilical cord blood; Flt3-L, Flt3 ligand; TPO, thrombopoietin; SCF, stem cell factor; CFU-GM, CFU-granulocyte-macrophage; BFU-E, burst-forming unit-erythroid; Meg-aggregate, megakaryocytic aggregate; ELTC-IC, extended long-term culture-initiating cell; LDA, limiting-dilution analysis; 7-AAD, 7-amino actinomycin-D.

Received for publication January 21, 2003. Accepted for publication July 16, 2003.

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