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TISSUE-SPECIFIC STEM CELLS

The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors

^aInstitut Cochin, Département d'Hématologie, Paris, France;
^bInstitut National de la Santé et de la Recherche Médicale, U567, Paris, France;
^cCentre National de la Recherche Scientifique, Unité Mixte de Recherche 8104 Paris, France;
^dFaculté de Médecine René Descartes, Université Paris, Unité Mixte 3, Paris, France;
^eFaculté de Médecine Paris-Sud 11, Université Paris-Sud, le Kremlin-Bicêtre, France;
^fInstitut National de la Santé et de la Recherche Médicale, U753, and
^gCentre d'Investigation Clinique Biothérapies, Institut Gustave Roussy, Villejuif, France

Key Words. Human hematopoiesis • Umbilical cord blood • Cell expansion • Lymphoid progenitors • HOXB4

Correspondence: Correspondence: Serge Fichelson, M.D., Ph.D., U567, Département d'Hématologie, Institut Cochin, Maternité Port-Royal, 123 Boulevard de Port-Royal, 75014 Paris, France. Telephone: 33-1-53-10-43-81; Fax: 33-1-43-25-11-67; e-mail: fichelson{at}cochin.inserm.fr

Received on September 4, 2007; accepted for publication on October 18, 2007.

First published online in STEM CELLS EXPRESS  October 25, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34⁺ hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The HOXB4 homeoprotein is an important regulator of hematopoietic stem cell (HSC) self-renewal and expansion. In mice, retrovirus-driven transduction of the hoxb4 gene coding sequence into adult HSCs and progenitors leads to a dramatic expansion of these cells without modifying their differentiation potentials. Importantly, no tumor process due to hoxb4 overexpression was ever reported [1, 2]. Furthermore, the ectopic expression of hoxb4 in primitive HSCs derived from murine yolk sac or embryonic stem cells allows these cells to acquire "adult-like" HSC characteristics, regarding both their phenotype and their contribution to lymphomyeloid hematopoietic reconstitution of irradiated mice [3]. Experiments performed in a model of nonhuman primates also demonstrated that hoxb4 is able to strongly expand simian HSCs, with a more pronounced effect on short-term repopulating cells [4]. The impact of hoxb4 on HSC expansion has also been documented in humans, but most reports stated that hoxb4-related expansion rates were less remarkable for human than for mouse HSCs [5, 6]. In a prospect of a further clinical use of HOXB4, we and others established that the HOXB4 protein itself was able to expand HSCs and myeloid progenitors ex vivo [7, 8]. However, the lymphoid and/or lymphomyeloid potentiality of hematopoietic progenitor cells (HPCs) after expansion by HOXB4 remain unclear. Although Schiedlmeier et al. reported that retrovirus-driven enforced overexpression of hoxb4 severely impaired the myeloerythroid and lymphoid differentiation of human CD34⁺ cells in vitro and in vivo [6], another study showed that ectopic constitutive expression of hoxb4 induced in vitro expansion of both myeloid and B-lymphocyte progenitors derived from human CD34⁺ cells [5]. Moreover, apart from a report of our group that deals with HOXB4 effects on natural killer (NK)-cell progenitors [9], the potential activity of the HOXB4 factor on the expansion of defined subpopulations of immature lymphoid or lymphomyeloid progenitors has not been described thus far.

In this report, we address the consequences of direct exposure of HPCs polarized toward the lymphoid lineages to the HOXB4 homeoprotein. Since homeoproteins can behave as cell-penetrating molecules in a passive and reversible internalizing process [10, 11], our group used the active secretion of HOXB4 protein by engineered MS-5 mouse stromal cells, the MS-5/signal peptide (SP)-HOXB4 cell line. Thus, we demonstrated that HOXB4 passively enters human hematopoietic cells and favors ex vivo expansion of HSCs without altering the myeloid differentiation potential of these cells in vitro or their myeloid and B lymphoid differentiation potentials in vivo [7, 12]. Moreover, we previously characterized umbilical cord blood (UCB)-derived CD34⁺CD45RA^hiCD7⁺ and CD34⁺CD10⁺Lin^– HPCs as early lymphoid progenitors polarized toward T-/NK- and B-cell lineages, respectively. We provided evidence for their in vitro independent emergence from CD34⁺CD45RA^intCD7^– lymphomyeloid progenitors and brought new insights into the physiological mechanisms by which these cells contribute to early lymphopoiesis [13, 14]. Here, we show that the presence of the HOXB4 homeoprotein in the cultures (a) results in dramatic expansion of B-, T-, and NK-cell progenitors when derived from more immature HPCs (CD34⁺CD38^lo/–), (b) specifically triggers expansion of the myeloid clonogenic progenitors derived from lymphomyeloid HPCs (CD34⁺CD45RA^intCD7^–), and (c) favors expansion of NK-cell progenitors and the maintenance of T-cell progenitors derived from T/NK lymphoid progenitor cells (CD34⁺CD45RA^hiCD7⁺). In contrast, the B-cell progenitors themselves (early CD34⁺CD10⁺Lin^– and late CD34⁺CD10⁺CD19⁺) are unaffected by the presence of HOXB4. Altogether, our results elucidate the effects of the HOXB4 homeoprotein in the early stages of human lymphopoiesis and should provide the basis for the development of new therapeutic strategies that include HOXB4-mediated expansion of human lymphoid progenitors.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Cell Lines
The mouse stromal cell lines MS-5, MS-5/SP-HOXB4 (MS-5 transduced with a lentiviral vector containing the mouse immunoglobulin -chain leader sequence for protein secretion upstream of the human hoxb4 cDNA), and MS-5/enhanced green fluorescent protein (EGFP) (MS-5 transduced with a vector containing the egfp cDNA, referred to as control) [7] were grown in complete -minimal essential medium containing 10% fetal calf serum (FCS) (Invitrogen, Cergy Pontoise, France, http://www.invitrogen.com).

Isolation and Immunolabeling of CD34⁺ Cells
Normal UCB units were collected according to institutional guidelines and after obtaining informed consent of the mothers. Following mononuclear cell separation (Lymphoprep; Fresenius Kabi, Sèvres, France, http://www.fresenius-kabi.com), CD34⁺ cells were enriched with the StemSep system (StemCell Technologies, Meylan, France, http://www.stemcell.com) according to the manufacturer's instructions. For isolation of HPC subpopulations, purified CD34⁺ cells were incubated for 30 minutes at 4°C in phosphate-buffered saline (PBS) containing 2% FCS, with CD34-fluorescein isothiocyanate (FITC), CD34-phycoerythrocyanin 5 (PECy5), CD34-allophycocyanin (APC) (clone 581), CD38-phycoerythrin (PE) (clone T16), CD7-FITC (clone 8H8.1) (all from Beckman Coulter, Villepinte, France, http://www.beckmancoulter.com), CD45RA-PE (clone HI100; BD Pharmingen, Le Pont de Claix, France, http://www.bdbiosciences.com), CD10-PECy5 (clone A1B1; Beckman Coulter), or CD19-PE (clone J4.119; Beckman Coulter) monoclonal antibodies (mAbs) diluted to a final concentration of 1:50. Then, HPCs were sorted, using a FACSVantage cell sorter (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com).

Primary Cocultures
As described [7], CD34⁺CD38^lo/– cells and CD34⁺CD45RA^intCD7^– HPCs (500–1,500 cells per cm²) were cocultured for 3 weeks with 30-Gy-preirradiated MS-5/SP-HOXB4 or MS-5/EGFP cells in standard complete H5100 human long-term culture medium (StemCell Technologies) in 25 cm² flasks [T25 flasks] (Dutscher, Brumath, France, http://www.dutscher.com). CD34⁺CD45RA^hiCD7⁺ HPC (400-800 cells per cm²), CD34⁺Lin^–CD10⁺ HPC, and CD34⁺CD10⁺CD19⁺ pro-B-cell (300–500 cells per cm²) cocultures were conducted for 2 weeks with both types of MS-5 cells. At the end of these cultures, total cells and CD34⁺ cells were counted to allow the determination of the expansion factor of total cells and CD34⁺ cells. CD34⁺ cells were further enriched to an average of 97% with the StemSep system prior to lymphoid and myeloid differentiation assays (Fig. 1B, steps I and II).

View larger version (41K): [in this window] [in a new window]   Figure 1. Fluorescence-activated cell sorting (FACS) isolation and culture procedure of umbilical cord blood CD34+ HPC subsets. (A): FACS analysis of HPCs according to CD34, CD38, CD45RA, CD7, CD10, and CD19 expression, before and after cell sorting (purity >98%); Lin– indicates CD7–CD19– [13, 14]. The various sorted cells are numbered i to v, according to the description in the first section of Results. (B): Schematic representation of the three steps of HPC expansion analysis. (C): Analysis of the presence of the HOXB4 protein within various HPCs subpopulations cocultured with either MS-5/signal peptide-HOXB4 (dark line) or MS-5/enhanced green fluorescent protein (gray histogram). Data are from one experiment out of two. Abbreviations: cy, cytoplasmic; FTOC, fetal thymic organotypic culture; HPC, hematopoietic progenitor cell; NK, natural killer.

5,6-Carboxyfluorescein-Diacetate-Succinimidyl-Ester Labeling
5,6-Carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) labeling of CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPCs was carried out as described previously [14–16]. Briefly, cells were incubated with 2.5 µM CFSE (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for 10 minutes at 37°C and then quenched with cold FCS. Labeled cells were then cocultured with MS-5/SP-HOXB4 and MS-5/EGFP as described above and analyzed at days 3, 5, and 7 of coculture using a FACSCalibur flow cytometer.

Assessment of NK- and B-Cell Differentiation Potentials
Sorted CD34⁺ cells derived from primary cocultures with either MS-5/SP-HOXB4 or MS-5/EGFP cells were cultured under NK- and B-cell differentiation conditions (Fig. 1A, steps II and III). For NK-cell culture conditions, CD34⁺ cells (1,000 cells per cm²) were cocultured for 3 weeks with unmodified MS-5 cells, in RPMI 1640 medium, 5% FCS (StemCell Technologies), 10% human AB serum (Jack Boy, Toronto, ON, Canada), and 0.1 mM β-mercaptoethanol (β-ME) with the following human recombinant cytokines: 50 ng/ml stem cell factor (SCF) (Amgen, Thousand Oaks, CA, http://www.amgen.com), 5 ng/ml interleukin (IL)-2, and 1 ng/ml IL-15 (Diaclone, Besançon, France, http://www.diaclone.com/anglais). B-cell differentiation of CD34⁺ HPCs was assessed by growing cells (10⁴ cells per cm²) for 3 weeks in unmodified MS-5 cell-coated plates in Iscove's modified Dulbecco's medium (Invitrogen) complemented with 5% FCS, 2 mM L-glutamine, and 0.1 mM β-ME, without cytokines. Half of the complete medium was replaced weekly.

Assessment of T-Lymphocyte Differentiation Potential
Fetal thymic organotypic cultures (FTOCs) were performed as reported [13, 14, 17]. Briefly, thymic lobes were collected from 15–17-day-old nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse embryos under a binocular magnifying glass. Hanging drops were prepared in Terasaki plates (Polylabo, Strasbourg, France, http://www.reactolab.ch) by adding 25 µl of RPMI 1640 medium containing 10% human AB serum with 3 x 10³ cells per lobe (at day 0) or total cells derived from 3-week cocultures of either 3 x 10³ or 1.5 x 10³ cells per lobe. Six to nine lobes were used for each condition. Plates were immediately inverted and incubated for 48 hours in a humidified incubator. Lobes were then removed, transferred onto floating Nucleopore filters (Millipore SA, Molsheim, France, http://www.millipore.com) in six-well plates, and cultured for 4 weeks. Lobes were subsequently removed and pooled or treated individually, and their cell contents were recovered by mechanical disruption, numbered, labeled, and analyzed with a FACSCalibur.

Immunolabeling of NK, B, and T Cells
For phenotypic analysis of cells cultured in lymphoid conditions, cells were incubated for 30 minutes at 4°C with the following mAbs (final concentration, 1:100) in PBS containing 2% FCS, washed, and then analyzed with a FACSCalibur: CD56-APC (clone N901) and CD3-FITC (clone UCHT1) (both from Beckman Coulter) for NK-cell detection; CD19-FITC (clone HIB19; BD Pharmingen) for B lymphocytes; and CD45-FITC (clone KC56), CD4-PECy5 (clone 13B8.2), CD8-APC (clone B9.11), and T-cell receptor β (TCRβ)-PE (clone BMA031) (all from Beckman Coulter) for T-cell detection. Isotype-matched FITC-, PE-, PECy5-, and APC-conjugated irrelevant mAbs were from BD Pharmingen and Beckman Coulter.

Clonogenic Assays
Five hundred CD34⁺ HPCs were seeded in 35-mm duplicate dishes (Dutscher) in 1 ml of standard Methocult GF+ H4100 medium (StemCell Technologies), supplemented with the following human recombinant cytokines: 2 U/ml erythropoietin, 15 ng/ml IL-3 (both from Kirin Brewery, Tokyo, http://www.kirin.com), 15 ng/ml SCF, 20 ng/ml granulocyte-colony stimulating factor (both from Amgen), and 5 ng/ml granulocyte-macrophage colony-stimulating factor (Schering-Plough, Kenilworth, NJ, http://www.sch-plough.com). Dishes were incubated at 37°C in a fully humidified atmosphere containing 5% CO₂. Erythroid burst-forming units (BFU-E) and granulocyte and/or macrophage colony-forming units (CFU-G/GM/M) were scored at day 15 under an inverted microscope.

Cell Expansion Analysis
The relative fold expansion corresponds to fold increase under MS-5/SP-HOXB4 conditions divided by fold increase under MS-5/EGFP conditions. Fold increase results from the absolute number of cells recovered per culture divided by that of day 0.

Statistical Analysis
To analyze differences in mean values between low-number samples, data were statistically tested using the Kruskal-Wallis or the Mann-Whitney test (in independent cases) and the rank-signed Wilcoxon test (in matched cases). Differences with p < .05 were considered statistically significant.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
HOXB4 Promotes the Expansion of Both Total and CD34⁺ Cells Derived from UCB CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ Subsets
To assess whether the passive transfer of HOXB4 protein can lead to the expansion of human lymphoid progenitors, we purified and studied five subpopulations of cells from UCB (Fig. 1A): (a) CD34⁺CD38^lo/– immature cells that comprise HSCs; (b) CD34⁺CD45RA^intCD7^– cells that exhibit weak lymphoid differentiation potential but are enriched in myeloid progenitors [13, 18]; (c) CD34⁺CD45RA^hiCD7⁺ and (d) CD34⁺CD10⁺Lin^– HPCs, both of which correspond to the immediate progenies of the CD34⁺CD45RA^intCD7^– cells and that we previously characterized as early T/NK and B lymphoid progenitor cells, respectively [13, 14, 19]; and (e) CD34⁺CD10⁺CD19⁺ pro-B cells.

Each of these five subpopulations of HPCs was used to establish long-term cocultures with MS-5/SP-HOXB4 cells and MS-5/EGFP cells as control (Fig. 1B, step I). We first verified that delivery of HOXB4 in the culture supernatant did lead to HOXB4 inflowing into each subset of tested HPCs (Fig. 1C). Then, we sought the presence of CD34⁺ progenitor cells within each coculture after 2–3 weeks of coculture (Fig. 1B, step II). The percentages of CD34⁺ cells obtained from CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPCs cocultured with MS-5/SP-HOXB4 and MS-5/EGFP control cells were 27.7% ± 5.4% and 24.9% ± 3.4%, 38.2% ± 8.6% and 38.3% ± 10%, and 20.9% ± 4.4% and 10.7% ± 4.5%, respectively, whereas CD34⁺CD10⁺Lin^– and CD34⁺CD10⁺CD19⁺ pro-B cells cocultured with MS-5/SP-HOXB4 versus MS-5/EGFP led to 1.8% ± 1.2% versus 4.2% ± 3.2% and 3.3% ± 0.5% versus 4.3% ± 2.2%, respectively (Fig. 2A). These results show that whatever the coculture (i.e., with or without HOXB4), it was possible to maintain a proportion of available CD34⁺ progenitors derived from each cell subpopulation at the exception of primary pro-B cells that could not efficiently be maintained in coculture. Numbers of total and CD34⁺ cells were also analyzed to establish both absolute and relative cell expansion rates, before studying the differentiation potentials of each subpopulation (Fig. 1B, steps II and III). CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPC cocultures with MS-5/SP-HOXB4 resulted in a dramatic overall enrichment in total cells and CD34⁺ cells by comparison with MS-5/EGFP control cocultures, as presented in Table 1. Indeed, after coculture of HPCs with MS-5/SP-HOXB4 cells, the relative expansion rates of total cells and CD34⁺ cells were, respectively, (a) 3.4 ± 0.9 and 3.2 ± 0.8 from CD34⁺CD38^lo/– cells, (b) 3.4 ± 0.5 and 3.4 ± 0.7 from CD34⁺CD45RA^intCD7^– HPCs, and (c) 3.3 ± 1 and 7.8 ± 2.5 from CD34⁺CD45RA^hiCD7⁺ HPCs (Fig. 2A, 2B). On the contrary, culture of CD34⁺Lin^–CD10⁺ and CD34⁺CD10⁺CD19⁺ primary pro-B cells did not result in any increase of cell production (Fig. 2B, 2C), although HOXB4 was detectable within these cells after a 24-hour coculture (Fig. 1C). Furthermore, these data suggest that HOXB4 did not modify the proportion of CD34⁺ cells among cocultures cells in comparison with control cocultures. Thus, the HOXB4 homeoprotein promotes the expansion of total cells and CD34⁺ cells derived from immature HPCs (CD34⁺CD38^lo/–), lymphomyeloid HPCs (CD34⁺CD45RA^intCD7^–), and T/NK HPCs (CD34⁺CD45RA^hiCD7⁺) but fails to sustain the expansion of cells derived from either CD34⁺CD10⁺Lin^– or CD34⁺CD10⁺CD19⁺ primary B progenitor cells. In the CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPCs cocultures, non-CD34⁺ cells corresponded to myeloid and B cells, whereas CD34^– cells derived from pro-B cell cocultures corresponded exclusively to CD10⁺CD19⁺ B cells (supplemental online Fig. 1). Taken together, these data demonstrate that HOXB4 preferentially targets immature CD34⁺CD38^lo/– cells [7], CD34⁺CD45RA^intCD7^– and CD34⁺CD45RA^hiCD7⁺ HPCs, which are endowed with certain multipotentiality [13]. To determine whether HOXB4 actually promotes growth or survival of CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPCs, analysis of cell division profile was performed on each cell population. Cell division kinetics during cultures was assessed using cell proliferation history by a high-resolution procedure based on the decrease of cell fluorescence after staining with CFSE (Fig. 3). CD34⁺ cells derived from cocultures of CD34⁺CD38^lo/–, CD34⁺CD45RA^intCD7^–, and CD34⁺CD45RA^hiCD7⁺ HPCs with MS-5/SP-HOXB4 displayed more divisions than those derived from control cocultures: as early as day 3, they produced at least one more generation of progeny in the presence of HOXB4 than control cultures. These data demonstrate the rapid proliferative response of these cells when exposed to HOXB4. Altogether, our data support the idea that HOXB4 rapidly activates the transcription of genes involved in the regulation of cell division [20].

View larger version (25K): [in this window] [in a new window]   Figure 2. Relative expansion of cells. (A): Percentages of CD34+ cells in cocultures of hematopoietic progenitor cells (HPCs) with either MS-5/signal peptide (SP)-HOXB4 () or MS-5/enhanced green fluorescent protein (EGFP) (). (B): Relative fold expansion of total cells. Cells were cocultured with MS-5/SP-HOXB4 () or MS-5/EGFP control cells (baseline 100%). (C): Relative fold expansion of total CD34+ cells. CD34+ cells were derived from HPCs cocultured with MS-5/SP-HOXB4 () or MS-5/EGFP control cells (designated as 100%). Results are presented as mean ± SEM; n = 5 for CD34+CD38lo/–, CD34+CD45RAintCD7–, and CD34+CD45RAhiCD7+ HPCs; n = 3 for CD34+CD10+Lin– and CD34+CD10+CD19+ pro-B cells. Significant differences were determined by the Kruskal-Wallis test for global comparison between independent samples (p < .01 [B]; p < .00001 [C]), and by the Mann-Whitney test for comparison between two independent groups: CD34+CD38lo/–, CD34+CD45RAintCD7–, CD34+CD45RAhiCD7+ versus CD34+CD10+Lin– and CD34+CD10+CD19+ HPCs (p < .0002 [B]; p < .00005 [C]).

View larger version (26K): [in this window] [in a new window]   Figure 3. Cell division profile. Kinetics of umbilical cord blood CD34+ cells during cocultures. Representative profiles of flow cytometry analysis of CFSE-labeled CD34+ cells derived from CD34+CD38lo/–, CD34+CD45RAintCD7–, and CD34+CD45RAhiCD7+ hematopoietic progenitor cells cocultured with MS-5/signal peptide-HOXB4 (dark line) or MS-5/enhanced green fluorescent protein (light line). The position of the cells that did not divide corresponds to the fluorescence intensity of the cells incubated with paraformaldehyde (dotted line). n corresponds to the number of cell divisions between day 0 undivided cells and the first peak obtained at day 3; the number above each peak thus represents division numbers according to the fluorescence intensity. Abbreviation: CFSE, 5,6-carboxyfluorescein-diacetate-succinimidyl-ester.

View larger version (24K): [in this window] [in a new window]   Figure 4. Relative expansion of total NK-, myeloid-, and B-cell progenitors. (A): Relative fold expansion of total NK progenitor cells. NK cells were derived from CD34+ cells purified from hematopoietic progenitor cells (HPCs) cocultured with MS-5/signal peptide (SP)-HOXB4 () or MS-5/enhanced green fluorescent protein (EGFP) control cells (designated as 100%) (mean ± SEM; n = 3). Significant difference was determined by the Mann-Whitney test by comparison between two independent groups: CD34+CD45RAintCD7– versus CD34+CD38lo/– or CD34+CD45RAhiCD7+ HPCs (p < .05). (B): Percentages of NK cells. NK cell cultures of CD34+ cells isolated from HPCs cocultured with either MS-5/SP-HOXB4 () or MS-5/EGFP () were analyzed for the expression of CD56 and CD3 (mean ± SEM; n = 3). (C): Relative fold expansion of total colony-forming cells (CFCs). CFCs were derived from CD34+CD45RAintCD7– HPCs cocultured with MS-5/SP-HOXB4 () or MS-5/EGFP control cells (designated as 100%) (mean ± SEM; n = 3). (D): Percentages of CFCs. Methylcellulose clonogenic cultures of CD34+ isolated from CD34+CD45RAintCD7– HPCs cocultured with either MS-5/SP-HOXB4 () or MS-5/EGFP () were analyzed for the presence of CFU-G/GM, CFU-M, and BFU-E. (E): Relative fold expansion of total B-progenitor cells. B lymphocytes were derived from CD34+ cells purified from CD34+CD38lo/– HPCs cocultured with MS-5/SP-HOXB4 () or MS-5/EGFP control cells (designated as 100%) (mean ± SEM; n = 3). (F): Percentages of B lymphocytes. B-cell cultures of CD34+ isolated from CD34+CD38lo/– HPCs cocultured with either MS-5/SP-HOXB4 () or MS-5/EGFP () were analyzed by fluorescence-activated cell sorting for the expression of CD19 (mean ± SEM; n = 3). Abbreviations: BFU-E, erythroid burst-forming units; CFU-G/GM, granulocyte and/or macrophage colony-forming units; CFU-M, macrophage colony-forming units; NK, natural killer.

HOXB4 Enhances the Number of B-Lymphocyte Progenitors Derived from CD34⁺CD38^lo/– Cells
Considering the scarcity of B-lymphocyte precursors among CD34⁺CD45RA^intCD7^– HPCs (approximately 1:195) (data not shown), and because HOXB4 did not promote direct expansion of primary B progenitors (Fig. 2), HOXB4-mediated expansion of B-lymphocyte progenitors was analyzed on cells derived from the CD34⁺CD38^lo/– primitive cell subset. Thus, three independent coculture experiments of CD34⁺CD38^lo/– cells with MS-5/SP-HOXB4 followed by secondary CD34⁺ cell cultures under B-cell differentiation conditions were performed and led to overall enrichment in total CD19⁺ B lymphocytes of 6.7- to 19-fold, whereas control cocultures with MS-5/EGFP led to only 0.5- to 10-fold expansion, resulting in a relative expansion rate of total B-lymphocyte progenitors of 10.8 ± 8.8 (Fig. 4E). Moreover, the B-cell differentiation potential of CD34⁺ cells derived from either coculture was equivalent, since no significant difference in the percentage of CD19⁺ B lymphocytes was observed (Fig. 4F). These data suggest that HOXB4 promotes the in vitro expansion of B-cell progenitors derived from CD34⁺CD38^lo/– cells without modifying their differentiation potential, in agreement with previous reports [5, 7].

HOXB4 Influences the Growth of T-Lymphocyte Progenitors Derived from CD34⁺CD38^lo/– and CD34⁺CD45RA^hiCD7⁺ Cells
To test whether HOXB4 also promotes the expansion of T-lymphocyte progenitors, we performed FTOCs with total cells derived from cocultures of CD34⁺CD38^lo/– immature cells and of CD34⁺CD45RA^hiCD7⁺ T/NK progenitors with MS-5/SP-HOXB4 and MS-5/EGFP cells and from the noncocultured day-0 HPC counterparts. T-cell differentiation potential of HPCs was evaluated by the emergence of double-positive (DP) CD4⁺CD8⁺ and/or total TCRβ⁺ thymocytes.

We first performed bulk cultures of T cells derived from cocultures (pools of 7–9 fetal thymic lobes per condition). The number of human thymocytes derived from CD34⁺CD38^lo/– cells was increased by sevenfold when cocultured with MS-5/SP-HOXB4 versus 3.5-fold when cocultured with MS-5/EGFP, compared with the number of thymocytes derived from day 0 nonexpanded cells (Fig. 5A), leading to a twofold relative expansion rate of total thymocytes (Fig. 5B). By contrast, the numbers of thymocytes collected from cocultures of CD34⁺CD45RA^hiCD7⁺ cells were lower compared with day 0 nonexpanded cells. This reduction reached 29-fold for thymocytes derived from control cocultures with MS-5/EGFP, whereas it was only 5.8-fold with MS-5/SP-HOXB4 (Fig. 5A). These data indicate that HOXB4 regulates the survival of thymocyte progenitors during cultures, since the number of thymocytes retrieved was five times higher in the presence than in the absence of HOXB4 (Fig. 5B). Thus, HOXB4 induces absolute and relative expansion of T-cell progenitors derived from CD34⁺CD38^lo/– cells, whereas it allows only limited maintenance of T-cell progenitors derived from the CD34⁺CD45RA^hiCD7⁺ T/NK HPCs. In addition, the T-cell differentiation potentials of the CD34⁺ cells derived from CD34⁺CD38^lo/– cells cocultured with MS-5/SP-HOXB4 versus MS-5/EGFP were equivalent (12% and 4.6% vs. 12% and 5.6% of DP and TCRβ⁺ thymocytes, respectively) (Fig. 5C). Moreover, HOXB4 strongly improved the long-term maintenance of T-lymphocyte progenitors derived from CD34⁺CD45RA^hiCD7⁺ cultures (6% vs. 0% of DP cells and 5.3% vs. 2.8% of TCRβ⁺ thymocytes, after coculture with MS-5/SP-HOXB4 vs. MS-5/EGFP, respectively) (Fig. 5B). Taken together, these data show that HOXB4 favors the expansion of T-lymphocyte progenitors derived from CD34⁺CD38^lo/– HPCs and contributes to the maintenance of the T-cell potential of the CD34⁺CD45RA^hiCD7⁺ T/NK-polarized HPCs (Fig. 5B), without influencing the T-cell differentiation ability of these various progenitors.

View larger version (37K): [in this window] [in a new window]   Figure 5. Expansion of total T-cell progenitors. (A): Absolute thymocyte numbers derived from 12,500 day 0 nonexpanded CD34+CD38lo/– (left) and CD34+CD45RAhiCD7+ (right) hematopoietic progenitor cells (HPCs) (gray columns) or 12,500 day 0 HPCs cocultured with either MS-5/SP-HOXB4 (black columns) or MS-5/enhanced green fluorescent protein (EGFP) control cells (open columns). (B): Relative fold expansion of total thymocyte progenitors. Thymocytes were derived from fetal thymic organotypic cultures (FTOCs) performed with CD34+CD38lo/– and CD34+CD45RAhiCD7+ HPCs cocultured with MS-5/SP-HOXB4 (filled columns) or MS-5/EGFP control cells (designated as 100%). Thymocytes correspond to double-positive CD4+CD8+TCRβ+/– and single-positive CD4+TCRβ+ cells. (C): Percentages of thymocytes. Fluorescence-activated cell sorting analysis of cells derived from FTOCs performed from day 0 fresh CD34+CD38lo/– and CD34+CD45RAhiCD7+ HPCs or directly derived from HPCs cocultured with either MS-5/SP-HOXB4 or MS-5/EGFP. Cells were analyzed for the expression of CD4, CD8, and TCRβ. T cells were gated on human CD45 cells (not shown). Data are from one experiment of two. Abbreviations: SP, signal peptide; TCR, T-cell receptor.

View larger version (22K): [in this window] [in a new window]   Figure 6. Analysis of T-cell precursor frequencies. Fetal thymic organotypic cultures were performed with cells derived from an equivalent day 0 number of hematopoietic progenitor cells (CD34+CD38lo/– and CD34+CD45RAhiCD7+ cells) cocultured with MS5-SP/HOXB4 or MS-5/EGFP, and fluorescence-activated cell sorting analysis was performed on single fetal thymic lobes. Positive thymic lobes for T-cell differentiation (+) indicate the presence of thymocytes (here, total TCRβ+ cells indicated in boxes within dot plots). Data are from one experiment of two. Abbreviations: EGFP, enhanced green fluorescent protein; SP, signal peptide; TCR, T-cell receptor.

	DISCUSSION

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CFSE labeling showed that HOXB4 hastened the target cells to enter the mitotic cycle as hoxb4-transduced bone marrow cells whose improved growth rate is stabilized and stays constant in long-term culture (Fig. 3) [24]. Such a phenomenon could be distinguished from an effect of HOXB4 on cell survival, since no difference was observed between the very weak proportions of apoptotic cells found in cocultures of HPCs with either MS-5/SP-HOXB4 or MS-5 EGFP control after 2 weeks of coculture (data not shown). As far as the expansion of lymphoid cell subpopulations is concerned, no significant difference was observed between the expansion rates of NK-cell progenitors derived from either CD34⁺CD38^lo/– immature HPCs or CD34⁺CD45RA^hiCD7⁺ T/NK-committed HPCs (Fig. 4A). Furthermore, the relative expansion rate of T-lymphocyte progenitors derived from the CD34⁺CD45RA^hiCD7⁺ cells was superior to that of T-cell progenitors derived from the CD34⁺CD38^lo/– cells (Fig. 5B). However, the comparison of the absolute expansion rates of T-lymphocyte and NK-cell progenitors derived from both cell populations showed that the presence of HOXB4, associated with the activity of the MS-5 stromal cells, resulted in a more remarkable effect on CD34⁺CD38^lo/– cells (supplemental online Table 1). This suggests that at the level of committed T/NK-progenitors, HOXB4 acts to rescue the production of T- and NK-cell progenitors after a long-term culture during which CD34⁺CD45RA^hiCD7⁺ T/NK HPCs become exhausted (Fig. 5A). Furthermore, in our hands, HOXB4 was ineffective on primary CD34⁺CD10⁺Lin^– and CD34⁺CD10⁺CD19⁺ pro-B cells (Fig. 2). Altogether these data support the idea that HOXB4 preferentially targets immature cells that have not undergone lineage commitment, which is consistent with previous observations suggesting that hoxb4, like other members of the hoxa and hoxb gene clusters, is preferentially expressed in primitive human CD34⁺ cells, whereas its expression is downregulated to a low or undetectable level in more mature blood cells of all lineages [25]. Moreover, our data are in agreement with reports concerning mouse HSC expansion mediated by hoxb4 transduction that does not lead to any alteration in the lymphoid and myeloid differentiation potential of HSCs in vivo [2, 3]. However, our findings are at odds with those of Schiedlmeier et al. [6] and Brun et al. [26], who reported, respectively, (a) that retrovirus-driven enforced overexpression of hoxb4 affects myeloerythroid and lymphoid differentiation of human CD34⁺ cells in vitro and in vivo [6], and (b) that adenoviral hoxb4 overexpression in HSCs mainly directs these cells toward myeloid differentiation rather than increasing their proliferation [26]. The main difference between these studies and ours is related to the experimental model: whereas the other studies used retrovirus- or adenovirus-mediated infections of cells, leading to constitutive strong overexpression of hoxb4 in the target progenitor cells, our system is based on the passive and sustained transduction of relatively low doses of the homeoprotein into cells. Furthermore, direct transduction of hoxb4 into human CD34⁺ cells with the same recombinant lentiviral vector induced a deleterious effect on the growth of human HSCs and progenitors, instead of an expansion (unpublished data). Therefore, the observed differences may be related to a dose-response effect of HOXB4, as previously suggested [6, 27, 28]. In particular, the range of concentrations of HOXB4, which mediates desired activities with concomitant minimal unwanted side effects (the so-called "therapeutic window"), may be reached in our experimental model to allow HPC and HSC expansion without altering their intrinsic differentiation potential. More generally, this dose-response phenomenon could be extended to other members of the HOX family, such as HOXA10. Indeed, a model of transgenic mice in which the human hoxa10 cDNA is driven by an inducible promoter recently showed that the induction of proliferation of HSCs by HOXA10 homeoprotein was dependent on its concentration: high levels of HOXA10 had no effect on HSC proliferation and led to both accumulation of erythromegakaryocytic progenitors and blockage in the erythrocytic and megakaryocytic development, whereas intermediate concentrations of HOXA10 induced an increase in the repopulating capacity of HSCs [29].

Besides our study, which refers to expansion of defined subsets of HPCs polarized toward the lymphoid lineage, reports that deal with ex vivo lymphoid progenitor expansion or maintenance in human mostly involve procedures that include CD34⁺ HPCs or CD34⁺CD38^– primitive hematopoietic cells cultured with or without stromal cells [21, 22, 30–33]. Whereas our experiments consisted of coculture of HPCs with MS-5 cells in long-term procedure in the sole presence of the HOXB4 homeoprotein, followed by secondary cultures in lymphoid conditions, Lewis et al. cultured CD34⁺CD38^– cells with or without contact with the AFT024 mouse fetal liver stromal cell line in medium supplemented with cytokines that also led, in secondary NK-cell culture, to an in vitro expansion of NK culture initiating cells [31]. However, compared with noncultured cells, the expansion rate of NK-cell progenitors was very inferior (approximately sixfold increase) to that which we obtained from the CD34⁺CD38^lo/– HPCs in our model. Furthermore, B- and T-culture-initiating cell assays were not performed in that system [31]. Although in our model of HPC expansion, HOXB4 does not modify the lymphoid or myeloid intrinsic differentiation potential of expanded HPCs in comparison with control cultures, the procedures described above do not provide comparison between the differentiation potentials of expanded or nonexpanded cells [21, 22, 30–32]. Furthermore, our report provides evidence for the expansion of NK-cell progenitors derived from CD34⁺CD38^lo/– HPCs and resulting fully functional mature NK cells [9]. Activated NK cells represent a convenient tool for therapy of certain hematopoietic malignant disorders, during or after allogenic bone marrow transplantation. NK cells are now recognized as important cytotoxic effectors involved in the graft versus leukemia [34] and in the maintenance of remission in a minimal residual disease setting after autologous transplantation [35].

Investigating the expression of hox genes during lymphoid development provides a useful approach to assess their contribution to early human lymphopoiesis. Thus, at the opposite of hoxb4, which is not substantially expressed in committed lymphoid progenitors [25, 36], other hox genes are expressed in immature HPCs and in cells of lymphoid lineages. In particular, expression of hoxc4 was ascertained in the CD34⁺CD38^lo/– cells and at subsequent stages of differentiation up to terminally matured B and T lymphoid cells [36, 37]. Furthermore, retroviral overexpression of hoxc4 induces in vitro expansion of very early HPCs as well as committed progenitors (unpublished data; Daga et al. [38]). Thus, we can propose an alternative strategy that includes the combination of both HOXB4 and HOXC4 homeoproteins for lymphoid progenitor expansion. Moreover, inefficient action of HOXB4 on the CD34⁺CD45RA^hiCD7⁺ T-cell precursors can lead to a search for other hox genes involved in T-cell development, such as hoxa10, whose high expression level in child thymus precursor cells is noticeable and follows that of hoxc4 [36]. CD34⁺CD45RA^hiCD7⁺ T-cell precursors that we characterized as fetal thymus colonizing cells [14] could thus be considered a candidate for T progenitor cell expansion by HOXC4 and/or HOXA10.

	CONCLUSION

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Our work elucidates the effects of HOXB4 on the ex vivo expansion of defined cell actors of early human lymphopoiesis. HOXB4 acts all the better since target cells are endowed with certain multipotentiality. Our findings provide evidence that HOXB4-mediated expansion of NK, B, and T progenitors derived from CD34⁺CD38^lo/– immature HPCs is a powerful alternative to classic lymphoid progenitor cell expansion procedures, thus supporting the potential use of this protein for stem cell expansion in a therapeutic setting.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported by grants from the Agence Nationale pour la Recherche, the Ligue Nationale contre le Cancer, the Ingénierie Assistance Conseil Presentation Informatique and Espace Direct groups. We thank Sylvie Gisselbrecht, Anne Caignard, and Béla Papp for helpful discussions; Micaël Yagello for flow cytometry cell sorting; Vivian Viallon for help in statistical analysis; and Azzedine Yacia for technical assistance. F.P. and I.V. contributed equally to this work.

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