Functional characterization of human CD34+ cells that express low or high levels of the membrane antigen CD111 (nectin 1)

Abstract

Nectins are recently described adhesion molecules that are widely expressed on many tissues, including the hematopoietic tissue. Nectin 1 (CD111) is expressed on a higher proportion of mobilized peripheral blood (mPB) than cord blood (CB) CD34+ cells, and of CD34+/CD38+ cells when compared with CD34+/CD38 cells. We studied functional properties of human CB and mPB CD34+ cells that express low or high levels of CD111. CD34+/CD111dim cells contain a higher proportion of cells in G0/G1 phase than CD34+/CD111bright cells. CD34+/CD111bright cells contain more erythroid progenitors: CFU-E, than their counterparts, which on the opposite contain more HPP-CFC. Limiting dilution analyses demonstrate a higher frequency of immature progenitors: cobblestone-area colony-forming cells, in CD34+/CD111dim than in CD34+/CD111bright cells. In vitro differentiation assays demonstrate a higher frequency of B-, T- and dendritic-cell precursors, but less NK-cell precursors in CD34+/CD111dim cells. Evaluation of engraftment in NOD-SCID mice shows that SCID repopulating cells are more frequent among mPB CD34+/CD111dim cells. Liquid culture of CD34+/CD111dim cells with erythropoietin shows that CD111 expression increases simultaneously with CD36, following CD71 and before glycophorin A expression. In conclusion, immature human hematopoietic progenitors express low levels of CD111 on their surface. During erythroid differentiation CD34+ cells acquire higher levels of the CD111 antigen.

Introduction

Hematopoiesis is a tightly regulated process during which mature and functional cells that belong to different lineages, are continuously renewed through differentiation from a small pool of ancestral cells termed ‘stem cells’. Thus, identification of stem cells and understanding of mechanisms that control their survival, proliferation and commitment are of the utmost importance. For several decades, identification of stem cells has relied on phenotype: expression (or lack of expression) of a number of membrane antigens characterize various stages of mammalian hematopoiesis, including the earliest stages. One of the most useful tools for these approaches has been the description of the CD34 membrane antigen1, and the production of numerous monoclonal antibodies (MoAb) directed at various epitopes of the molecule. However, functional assays proved that the human CD34+ cell population was heterogeneous, containing both early and late progenitors; thus, investigators worked at refining their phenotypic analyses by subsetting the CD34+ cell population, using additional markers such as CD38,2,3,4 CD90 (Thy1),5,6 or the absence of antigens that are expressed following commitment to hematolymphoid lineages (‘lineage –‘).7

In some instances, the function of membrane antigens that are otherwise used as ‘stem cell markers’ is partially known. Some of these molecules are receptors for cytokines: this is the case for c-kit ligand or flt3 ligand. Others are molecules involved in cell-to-cell or cell-to-matrix interactions, or adhesion molecules. This has been suspected, although not formally demonstrated, for CD34 expressed on hematopoietic progenitors. Other adhesion molecules are clearly involved in the migration of progenitors to (‘homing’) or from (‘mobilization’) the bone marrow compartment: this is the case for VLA-4,8,9,10 PECAM-111 and LFA-1.12 Chemokines and their receptors such as SDF-1/CXCR48,13 and probably somatostatin/SSTR14 are also indispensable partners in these phenomena. Within the marrow, adhesion molecules may contribute to the proliferation, survival and differentiation of hematopoietic stem and progenitor cells, through interactions with their environment.15,16,17,18,19,20,21

Nectins are a recently described family of molecules that are structurally related to the immunoglobulin superfamily. Nectins were initially identified because of homology with the poliovirus receptor.22,23 Currently, four nectins have been described in humans.23,24,25,26 Since their description,22,23 expression and function of human nectin 1 or CD111, and nectin 2 or CD112 have been studied in a variety of models and tissues.24,27,28,29 Both molecules are widely expressed on numerous tissues of different embryonic origin, and appear to function as receptors for herpes simplex viruses.27,28 However, the physiological functions of these molecules have not been elucidated yet. The association between mutations in the nectin 1 gene and cleft lip/palate ectodermal dysplasia has been described.30 Nectin 1 is involved with nectin 3 in the formation of neurological synapses.29 Knock-out mice lacking nectin 2 have aberrant spermatozoa, and are sterile.31 The most plausible hypothesis is that nectins play a role in cell–cell interactions and recognition. Recent data suggest that nectins are involved in the tri-dimensional organization of diverse tissues including epithelium and endothelium, as they are expressed at the adherens junctions, on the lateral side of epithelial cells,26,32 and they are capable of homophilic or heterophilic interactions.26,33 In addition, nectin 2 could play a role in interactions between hematopoietic and endothelial cells, as it is expressed on both cell populations.24

In the present report, we describe the expression of CD111 (nectin 1) on human cord blood (CB) and adult mobilized peripheral blood (mPB) CD34+ cells. We next describe functional properties of human CD34+ cells that express low or high levels of CD111, using a variety of in vitro and in vivo assays.

Patients and methods

Patients

Following informed consent, apheresis samples were obtained from patients with a variety of poor-prognosis cancers, who underwent mobilization with rhG-CSF (Neupogen, Amgen Thousand Oaks, CA, USA, or Granocyte, Chugai, Paris, France) alone or in association with chemotherapy. Two samples were obtained from healthy donors for allogeneic transplantation. CB samples were obtained after delivery from informed mothers.

For all samples, mononuclear cells were enriched and red blood cells were depleted through density gradient separation (d=1.077 g/ml, Nycomed, Oslo, Norway). Adherent cells were depleted, and CD34+ cells were immunoselected, using the MACS technique according to the manufacturer's recommendations (Miltenyi Biotec, Bergisch-Gladbach, Germany).

MoAb and flow cytometry studies

MoAb used for this study are listed in Table 1. All analyses were conducted with a FACScalibur (Becton-Dickinson Immunocytometry Systems, BDIS, San Jose, CA, USA). Sorted cell populations were obtained with a FACSVantage (BDIS) equipped with a 448 nm argon laser.

Table 1 MoAbs used in these experiments

Cell cycle analyses

The percentages of cells in G0/G1, S and G2/M phases were evaluated for CD34+/CD111dim and CD34+/CD111bright cells, by staining living cells with propidium iodide. A total of 5 × 104–105 cells were fixed in 70% ethyl alcohol (Carlo Erba Reagenti, Milan, Italy) for 15 min, then treated with 200–300 μl of a 10 μg/ml DNAse-free RNAse solution (Roche Diagnostics Corporation, Indianapolis, IN, USA) for 15 min at 37°C. An equal volume of a 50 μg/ml propidium iodide solution was added, and cells were incubated for 1 h at 4°C before flow cytometry analysis.

Clonogenic cultures

The technique for the detection of granulomacrophagic progenitors (CFU-GM), erythroid progenitors (BFU-E and CFU-E) and early and highly proliferative progenitors (high-proliferative potential colony forming cells, HPP-CFC) has been described elsewhere.34,35,36Briefly, for CFU detection, 250 cells were seeded in triplicate in methyl-cellulose medium for 14 days in the presence of a combination of cytokines: IL3 (a kind gift from Amgen, Thousand Oaks, CA, USA), IL6 (Amgen), GM-CSF (Leucomax®, Novartis, Rueil-Malmaison, France), G-CSF (Amgen) at 10 ng/ml, erythropoietin, Epo (Eprex®, Janssen-Cilag, Issy-les-moulineaux, France) at 2 U/mL and SCF (Amgen) at 100 ng/ml. Colonies of at least 50 nonhemoglobinized cells were scored as CFU-GM. CFU-E contained one or two colonies of hemoglobinized cells. BFU-E contained at least three colonies of hemoglobinized cells. For HPP-CFC detection, 1000 cells were seeded in triplicate in an agar medium supplemented with a combination of cytokines: IL3, IL6, GM-CSF, G-CSF and flt3-L (R&D Systems, Minneapolis, MN, USA) at 20 ng/ml, and SCF at 100 ng/ml. HPP-CFC were scored on day 21.

Limiting dilution analysis of Cobblestone-area colony-forming cells (CAFC)

The technique was adapted from a previously published report.37 Monolayers of the MS-5 stromal cell line were established in 96-well plates, and the cells were allowed to reach confluence.38 Serial dilutions of CD34+/CD111dim and CD34+/CD111bright cells were then seeded on the monolayers. IMDM medium supplemented with 10% fetal calf serum (FCS, Stem Cell Technologies) was used for establishing monolayers, and for culturing hematopoietic progenitors; no human recombinant cytokine was added. CAFCs were scored at week 5.

In vitro differentiation in erythroid cells

CD34+/CD111dim cells from either mPB or CB were cultured at a concentration of 2 × 104 cells/ml, in serum-free medium: IMDM (BioWhitaker, Verviers, Belgique), supplemented with 20% BIT (Stem Cell Technologies Vancouver, BC, Canada), and either with a combination of Epo (2 U/ml), IL3 and SCF (10 and 100 ng/ml, respectively) to induce erythroid differentiation, or with a combination of IL3, IL6, G-CSF and GM-CSF at a concentration of 10 ng/ml for each, and SCF (100 ng/ml) to induce granulomonocytic differentiation. Cultures were maintained for 15 days at a temperature of 37°C, 5% CO2 in a humidified atmosphere and the medium was changed at day 7.

B- and NK-cell differentiation during coculture with the murine stromal cell line MS-5

CD34+/CD111dim and CD34+/CD111bright cells were cocultured with the murine stromal cell line MS-5, as previously reported;39,40 however, all exogenous cytokines were withdrawn to favor B-cell differentiation,41 in IMDM medium supplemented with 5% FCS, 5% human male AB serum (Institut Jacques Boy SA) and 10−4M of β-mercaptoethanol (Sigma, Steinheim, Germany). In separate experiments, 20 ng/ml IL15 (R&D Systems) and 50 ng/ml SCF were added to the cultures to favor NK-cell differentiation in IMDM medium supplemented with 15% FCS.

Dendritic-cell differentiation

CD34+/CD111dim and CD34+/CD111bright cells were cultured in 24-well plates at a concentration of 2 × 104 cells/ml in RPMI-1640 medium supplemented with 10% FCS, 20 ng/ml of SCF, 100 ng/ml of GM-CSF, 2.5 ng/ml of TNFα (R&D Systems) and 20 ng/ml of flt3 ligand at 37°C in a fully humidified incubator with a 5% CO2 atmosphere during 14 days.42 The culture medium was changed at day 7.

Fetal thymus organotypic cultures

Organotypic cultures were performed as previously described.39 In brief, thymus of 14–16 days old NOD-SCID mice embryos were dissected. CB CD34+ cells were transferred to thymic lobes using the hanging drop method in Terazaki plates (in our hands, mPB CD34+ cells did not differentiate towards the T-cell lineage in these conditions). In total, 10 000–20 000 cells in 25 μl of culture medium were put in each well. A complete culture medium containing 5 ng/ml of IL2 (Chiron, Suresnes, France), 20 ng/ml of IL7 (R&D systems), and 50 ng/ml of SCF was used. After a 48 h incubation in a humidified incubator at 37°C and 5% CO2, lobes were transferred onto floating filters (Isopore membrane, 25 mm diameter, 8 μm pore size; Millipore SA, St-Quentin-en-Yvelines, France) in a medium without cytokines, and with the same atmospheric conditions. Cells were recovered by mechanical disruption of thymic lobes after 30 days, and used for cytometric analysis.

Detection of NOD-SCID repopulating cells

The technique has been described elsewhere,40 and reproduced from previously published reports,39,43,44 but was nevertheless adapted to assay human adult peripheral blood progenitors. Briefly, NOD-SCID mice45 were obtained from Dr John Dick, Department of Molecular and Medical Genetics, Hospital for Sick Children, Toronto, Canada. Founders were bred at our animal facility. All animals were maintained in germ-free conditions. Animals, 6–9 weeks old, were used for xenotransplantation. Recipients were prepared with total body gamma irradiation at a dose of 350 cGy. Within 24 h of irradiation, mice were injected with 0.9–4 × 106 mPB CD34+/CD111dim cells or CD34+/CD111bright cells via the tail vein. After 10–12 weeks, animals were killed; bone marrow cells were recovered from femurs and tibia by flushing, and the spleen was mechanically disrupted. Mononuclear cells were enriched and red blood cells were depleted through density gradient separation and then analyzed by flow cytometry for the expression of human antigens.

Statistics

Descriptive statistics, comparisons of paired values with nonparametric Wilcoxon's test, and comparisons of unpaired values with Student's t-test were carried out by using the SPSS software for Windows (SPSS Inc., Chicago, IL, USA). A P-value of 0.05 was chosen for statistical significance.

Results

Phenotypic analyses of CD111 expression on human CB and adult mPB CD34+ cells

CD34+ cells obtained by immuno-selection from CB or apheresis samples (mean purity: 85±11% for CB, n=31; 80±22% for mPB, n=61) were simultaneously stained with a MoAb to CD34 and a MoAb to CD111. When compared to an isotype-matched irrelevant control, the CD111 expression pattern within the CD34+ cell subset does not discriminate a clearly positive population, but rather suggests a continuum of expression (Figure 1); for this reason, CD34+ cells will further be subset in CD34+/CD111bright and CD34+/CD111dim cells (see below). However, comparison of the CD34+ cell subpopulation from adult mPB and from CB shows that a higher percentage of cells in the former population displays a fluorescence above the threshold set up with the control antibody (49.3±21.6% vs 18.4±8.5%; P<0.001 with a Student t-test). Hence, the CD34+/CD111bright subset that accounts for approximately one-third of CD34+ cells represents cells with the highest level of CD111 expression in samples from adult mPB, and all CD111+ cells in samples from CB origin. The CD34+/CD111dim subset was arbitrarily defined as the equivalent subset of CD34+ cells that had the lower fluorescence for CD111 expression; thus, a population exists with an intermediate level of fluorescence for CD111: this population was not sorted (Figure 1b and c).

Figure 1
figure1

CD111 expression profile on CD34+ cells obtained by immunoselection from apheresis or CB samples. (a) CD111 expression profile on mPB CD34+ cells. (b) Sorting windows are indicated. CD34+/CD111dim cells and CD34+/CD111bright cells represent approximately one-third of all CD34+ cells for apheresis samples. (c) For CB samples, the CD34+/CD111bright subset contains all cells that display a fluorescence above the threshold set up with an irrelevant matched isotype control.

Further, we studied CD111 expression on CD34+/CD38+ and CD34+/CD38 cells, the latter being a minor subset (1.6±0.7% for mPB, 6.6±3.2% for CB) enriched with the most immature human progenitors. The percentages and mean fluorescence intensities (MFI) for CD111 expression were lower in the CD34+/CD38 than in the CD34+/CD38+ subset, both in cells originating from adult mPB (32±26.9% vs 63±16.8%, P<0.05, n=12; MFI: 31.3 vs 78.5) and from CB (10±18.4 vs 36±30.5%, P<0.05, n=11; MFI: 34 vs 69.1).

It has been demonstrated that CD111 behaves as an adhesion molecule through hetero- and homotypic interactions with members of the nectin family. Thus, we studied with multicolor flow cytometry the coexpression of other known adhesion molecules on CD34+/CD111+ and CD34+/CD111cells. Figure 2 demonstrates that the pattern of expression for CD11a (LFA-1), CD15 s, CD54 (ICAM-1), CD62L (L-selectin), CD49d (VLA-4), CD49e (VLA-5) and CD102 differs within the two subsets. CD49e, CD49d and CD102 are statistically significantly more expressed on CD34+/CD111+ cells than on CD34+/CD111 cells, although the differences are biologically small for CD49d and CD102. The opposite is observed with CD54, CD62L, CD15 s and CD11a which are significantly more expressed on CD34+/CD111 cells. These observations are valid for cells of mPB or CB origin.

Figure 2
figure2

Expression of a variety of adhesion molecules following CD111 expression. Expression of various adhesion molecules was determined on CD34+/CD111+ cells and CD34+/CD111 cells from mPB or CB. Results are means ±s.e.m. of six to 12 experiments. *P<0.05 using a Wilcoxon's test for paired samples.

Cell cycle analyses

Owing to the relation between cell cycle activity and stem cell potential within the hematopoietic progenitor cell population,4 we next determined the percentage of cells in G0/G1 phase in the two cell subsets: CD34+/CD111bright and CD34+/CD111dim. Table 2 demonstrates that the proportion of cells that are not actively cycling (G0/G1 phase) is higher in the CD34+/CD111dim population than in its CD34+/CD111bright counterpart. These observations are valid for mPB as well as CB cells.

Table 2 Cell cycle analysis

The frequency of clonogenic progenitors differs between CD34+/CD111dim and CD34+/CD111bright cells

Overall, the frequency of committed clonogenic progenitors, as estimated in methyl-cellulose assays, is higher in CD34+/CD111bright cells than in CD34+/CD111dim cells, although the difference appears to be statistically significant only for samples of mPB origin (Table 3). The higher frequency of mature erythroid progenitors (CFU-E) accounts for most of the difference; the frequency of BFU-E being equivalent in both cell subsets. In CB, the frequency of CFU-GM was significantly higher in the CD34+/CD111dim subpopulation.

Table 3 Clonogenic potential of CD34+/CD111bright and CD34+/CD111dim cells from adult mPB and CB

On the opposite, the frequency of more immature clonogenic progenitors – namely HPP-CFC – as assayed in agar cultures, was significantly higher in mPB or CB CD34+/CD111dim cells than in CD34+/CD111bright cells (Table 3).

CAFC are more frequent in CD34+/CD111dim cells than CD34+/CD111bright cells

Cells that are able to reinitiate hematopoiesis in culture systems with stromal cells, either long-term culture initiating Cells (LTC-IC) or CAFC, represent another index of stem cell activity. Using a limiting dilution assay with the murine stromal cell MS-5, we measured the frequency of CAFC37 in the two subpopulations. Despite intersample variability, Table 4 demonstrates the higher frequency of CAFC in the CD34+/CD111dim population of mPB origin; however, the frequency of CAFC appeared to be equivalent in the two subsets, for the three CB samples that we studied.

Table 4 Limiting dilution analyses of the frequency of CAFC in CD34+/CD111bright and CD34+/CD111dim cells

Evaluation of CD34+/CD111dim and CD34+/CD111bright cell differentiation capacity into B, T, NK and dendritic cells

Looking at the in vitro differentiation potential of the two subsets, we cocultured the two sorted cell populations with the murine stromal cell line, MS-5, in the presence or absence of a combination of IL-15 and SCF, to favor NK- or B-cell differentiation, respectively. NK and B cells were identified with phenotypic features. After 5 weeks of culture, mPB CD34+/CD111dim cells yielded more CD19+ but less CD56+ cells than CD34+/CD111bright cells. CB CD34+/CD111bright also produced more CD56+ cells, while numbers of CD19+ cells that derived from both CB subpopulations appeared to be equivalent (Table 5).

Table 5 Evaluation of CD34+/CD111dim and CD34+/CD111bright cell differentiation capacity into B, T, NK and dendritic cells

CD34+/CD111bright and CD34+/CD111dim were also cultured in conditions that were previously demonstrated to favor dendritic-cell differentiation;42 these cells were identified as CD1a+ cells, and further studied for the expression of costimulatory molecules: CD80, CD83 and CD86. At day 14, the percentage of CD1a+ cells was higher in cultured CD34+/CD111dim cells than in CD34+/CD111bright cells, either from mPB or CB origin (Table 5). This higher percentage translated into a higher absolute number of dendritic cells produced from mPB CD34+/CD111dim cells. Within the CD1a+ subset, the expression of CD80, CD83 and CD86 was comparable in cells obtained from the two cultured populations.

Finally, for the assessment of our two population ability to differentiate into T lymphocytes, we seeded CB CD34+/CD111dim and CD34+/CD111bright into NOD-SCID mouse thymic fragments, as previously described39. After 14 days of culture, the numbers of double positive CD3+/CD4+/CD8+ and the MFI for CD4 expression were higher in organotypic cultures initiated with the CD34+/CD111dim subset than in organotypic cultures initiated with its CD34+/CD111bright counterpart (three separate experiments; Table 5 for percentages and data not shown for MFI).

Evaluation of CD34+/CD111dim and CD34+/CD111bright cell ability to repopulate hematopoietic organs in NOD/SCID mice

CD34+/CD111dim and CD34+/CD111bright cells sorted from apheresis samples were infused in equal numbers via the tail vein into the bloodstream of sublethally irradiated NOD-SCID animals. Mice were killed at designated time points after injection, and the presence of cells of human origin was assayed by flow cytometric identification of cells that stained positive for human CD45 or CD71 in the bone marrow and the spleen.46 The presence of SCID repopulating cells (SRC) in CB CD34+/CD111dim and CD34+/CD111bright cells was not assessed, because of low cell numbers. Human cells were detected in both groups of animals. However, the percentage of CD45+ cells was higher when mice were engrafted with CD34+/CD111dim cells, than when mice were engrafted with CD34+/CD111bright cells (Table 6). Only small numbers of CD45/CD71+ cells–a phenotype that identifies the erythroid progeny of SRC – were detected in hematopoietic organs of engrafted animals, and these figures were similar for CD34+/CD111dim and CD34+/CD111bright cells. The distribution of CD45+ cells in myelomonocytic (CD13+/CD33+), megakaryocytic (CD41+) or B-lymphoid (CD19+/CD34) cells was similar in animals engrafted with CD34+/CD111dim or with CD34+/CD111bright cells (data not shown).

Table 6 Engraftment of sublethally irradiated NOD-SCID mice with mPB CD34+/CD111dim and CD34+/CD111bright cells

In vitro differentiation in erythroid cells

The high frequency of CFU-E in the CD34+/CD111bright subset suggested that CD111 may be highly expressed in the erythroid lineage, and prompted us to follow CD111 expression during in vitro differentiation. For this purpose, CD34+/CD111dim cells were sorted and cultured for 14 days in the presence of a cytokine combination known to favor erythroid differentiation, particularly as it contains Epo (SCF+IL3+Epo); as a control, the same cells were cultured in parallel with another cytokine combination that does not contain Epo, and rather favors myelomonocytic differentiation (SCF + IL3 + IL6 + GM-CSF + G-CSF). Figure 3 illustrates the kinetics of CD111 expression in one representative experiment for each source of cells; while CD111 was upregulated in both culture systems, conditions allowing for erythroid differentiation resulted in a higher percentage for CD111; in addition the MFI was also higher, suggesting higher numbers of the molecules on the cell surface (157 for the SCF+IL3+Epo condition vs 36.5 for the SCF+IL3+IL6+GM-CSF+G-CSF condition for mPB cells; 72 vs 46 for CB cells). Upregulation of CD111 expression appeared to occur later than CD71, synchronously with CD36 and prior to the appearance of glycophorin A, a marker that is strictly specific of the erythroid lineage. Of note, CD111 was only transiently upregulated, as its expression peaked and then decreased during the second week of culture. This pattern was similar for cells from mPB or CB origin.

Figure 3
figure3

CD34+/CD111dim cells from mPB samples or CB samples were cultured with two cytokines combinations allowing: erythroid differentiation (2 U/ml of Epo, 10 ng/ml of IL3 and 100 ng/ml of SCF) or myelomonocytic differentiation (IL3, IL6, G-CSF and GM-CSF at a concentration of 10 ng/ml and SCF at 100 ng/ml). Cultures were maintained for 15 days, and the expression of CD111, CD36, CD71, glycophorin A and CD34 on the cells was assessed at different time points.

Discussion

We demonstrate here that human CD34+ cells from CB or adult mPB origin are heterogeneous in terms of CD111 expression. The profile of expression for CD111 appears to be different from the profile of expression for CD112 (PRR2 or nectin 2),24 a structurally related surface antigen, and another member of this family of molecules; CD112 is found highly expressed on the vast majority of bone marrow CD34+ cells, an observation that is also valid for CB and mPB cells (data not shown). The pattern for CD111 shows a lower level of expression, and does not subset CD34+ cells into a truly negative and a truly positive population; it suggests that CD111 is expressed at various levels on most CD34+ cells. CB and mPB CD34+ cells express CD111 with different intensities; therefore, the gating for the CD34+/CD111bright cells may define slightly different populations when working with apheresis or CB samples, which may account for some of the different results observed when this subpopulation was studied with functional assays (for example, the lack of difference for CAFC frequency in CB CD34+/CD111dim cells and CD34+/CD111bright cells).

Nevertheless, all our observations suggest that CD111 is expressed at low levels during early stages of human hematopoietic differentiation. First, CD34+/CD38 cells, a minor subset of CD34+ cells known to be enriched for the most immature hematopoietic progenitors, express lower levels of CD111 than CD34+/CD38+ cells. Second, cell cycle analyses suggest a higher percentage of noncycling cells within the subpopulation that express low levels of CD111. Third, functional assays that detect early progenitors (HPP-CFC, CAFC and SRC) suggest that the frequency of such progenitors is higher within the CD34+/CD111dim cells than within the CD34+/CD111bright subset; however, the observation that HPP-CFC, CAFC and SRC can also be detected, albeit at lower frequencies, within the CD34+/CD111bright subpopulation, along with the relative frequency of CD34+/CD111dim cells, suggests that low expression of CD111 will not constitute an appropriate ‘human stem cell marker’ by itself.

In vitro differentiation assays suggest that B-, T- and dendritic-cell precursors are more frequent within the CD34+/CD111dim cells than within the CD34+/CD111bright cells, as more CD19+ cells, more CD3+/CD4+/CD8+ and more CD1a+ cells can be produced from CD34+/CD111dim cells than from equal numbers of CD34+/CD111bright cells. Surprisingly, NK-cell precursors were detected at a higher frequency in the CD34+/CD111bright subset; although a common origin has been reported for human T, B, NK and dendritic cells47, restriction in differentiation capacities may occur sequentially48 rather than simultaneously, and could be associated with different patterns of CD111 upregulation. Differentiation towards the nonlymphoid lineages suggests that cytokine combinations that induce erythroid differentiation (and contain Epo) also induce a strong upregulation of CD111 expression on the cell surface. This is compatible with the high numbers of CFU-E that were detected while culturing CD34+/CD111bright cells in methyl-cellulose assays, and with the increased expression of CD49d and CD49e on CD34+/CD111bright cells,49 as well as with the increased expression of CD11a on CD34+/CD111dim cells.50 The kinetics of CD111 expression, compared with the kinetics of expression for other useful markers of the erythroid lineage (CD71, CD36 and glycophorin A) suggests that CD111 may be highly upregulated during the transition from the BFU-E stage, to the CFU-E stage;50,51 during erythroid differentiation in vitro, CD111 expression appears to peak after approximately 1 week of culture, to decrease later. These observations are consistent with a previous report that demonstrates CD111 expression on mature (glycophorin A+) cells, with a higher percentage of glycophorin A+/CD111+ cells in the bone marrow than in peripheral blood.52

The role that CD111 may play during hematopoietic differentiation, and particularly during erythroid differentiation, is unknown. E-cadherin provides an example of an adhesion molecule, whose modulation can affect the ability of hematopoietic progenitors to differentiate towards the erythroid lineage: blocking antibodies directed to E-cadherin reduces the erythroid differentiation of human bone marrow mononucleated cells in vitro;53 in contrast, addition of a MoAb known to block CD111-mediated heterophilic interactions,54 failed to block the erythroid differentiation of human mPB CD34+ cells in vitro (the pattern of glycophorin A upregulation on cultured cells did not change), while we were able to reproduce inhibition with an anti-E-cadherin MoAb (data not shown). Elucidation of the role of CD111 during eythroid differentiation will deserve further investigation.

The function of nectin 1 and structurally related molecules remains elusive. Some members of the family act as virus receptors,55 and abnormal gene expression lead to defect in tissue organization.30,31 The localization of CD111 on the basolateral side of epithelial cells and endothelial cells, and associations of nectins with other membrane or cytoplasmic molecules in supra-molecular complexes leads to speculate that nectins may participate in the tri-dimensional organization of these tissues; however, the architecture of the hematopoietic tissue is poorly defined when compared to epitheliums, even though a stroma constituted of a variety of hematopoietic and nonhematopoietic cells embedded in an extracellular matrix exists, and is believed to play important roles in promoting the survival, proliferation and differentiation of hematopoietic stem and progenitor cells. Description of ‘anatomic’ structures has been limited to the ‘erythroblastic island’.56,57 Several adhesion molecules are likely to play a role in macrophage–erythroblast cell interactions.58,59 Whether CD111 also plays a role in the formation of these structures, and whether natural ligands for CD111 (nectins 3 and 4) are expressed on stromal cells, will deserve further attention, working with cells of marrow origin.

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Acknowledgements

This work was supported in part by Institut Paoli-Calmettes, and by a grant from the Comité Leucémie de la Fondation de France (# 990038) to CC. GB is the recipient of a grant from a joint research training program established by the French and Algerian governments. We thank all personnel at the Tumor Cell Collection (Biothéque) and at the Centre de Thérapie Cellulaire et Génique for access to apheresis and cord blood samples. We also thank Rémy Galindo at the Flow Cytometry Facility, and Patrick Gibier at the animal facility for their help during the conduct of these studies.

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Belaaloui, G., Imbert, AM., Bardin, F. et al. Functional characterization of human CD34+ cells that express low or high levels of the membrane antigen CD111 (nectin 1). Leukemia 17, 1137–1145 (2003). https://doi.org/10.1038/sj.leu.2402916

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Keywords

  • hematopoiesis
  • stem cells
  • adhesion moleculeserythroid differentiation

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