HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 2006-0321v1 25/6/1571    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lavenu-Bombled, C. Articles by Dubart-Kupperschmitt, A. Search for Related Content PubMed PubMed Citation Articles by Lavenu-Bombled, C. Articles by Dubart-Kupperschmitt, A.

TECHNOLOGY DEVELOPMENT

Glycoprotein Ib Promoter Drives Megakaryocytic Lineage-Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self-Inactivating Lentiviral Vector

^aInstitut Cochin, Département d'Hématologie, Paris, France;
^bInstitut Cochin, Plateforme de Microscopie Électronique, Paris, France;
^cInstitut National de la Santé et de la Recherche Médicale, U567, Paris, France;
^dCentre National de la Recherche Scientifique, Paris, France;
^eUniversité Paris Descartes, Paris, France;
^fService d'Hématologie Biologique, Hôpital Henri Mondor Université Paris XII, Créteil, France

Key Words. Megakaryocytes • Lentiviral vectors • Promoter • Human CD34+ cells • Gene transfer • Gene expression • Platelet Glycoprotein Ib

Correspondence: Anne Dubart-Kupperschmitt, M.D., Ph.D., Institut Cochin, Department of Hematology, Hôpital de Port-Royal, 123 Bd de Port-Royal, Paris 75014, France. Telephone: 33-153104369; Fax: 33-143251167; e-mail: dubart{at}cochin.inserm.fr

Received May 26, 2006; accepted for publication March 12, 2007.
First published online in STEM CELLS EXPRESS   March 22, 2007.

	ABSTRACT

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

 
Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34⁺ hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34⁺ cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIb, GpIIb, or EF1 gene regulatory sequences. Both GpIb and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIb promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIb gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34⁺ hematopoietic cells.

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

	INTRODUCTION

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

 
Hematopoietic cells are attractive targets for cell/gene therapy approaches. Moreover, targeting hematopoietic stem cells (HSC) opens the opportunity to ensure the life-long persistence of the transgene. Indeed, HSC have been used for a while in cell therapy and have proven their efficacy to permanently renew the circulating blood cell compartment with differentiated and fully mature cells. Efficient gene transfer in HSC, even though these cells are rare and slow-dividing, is now possible using new generations of self-inactivating lentiviral vectors, which also allow lineage-specific transcription of the transgene from an internal promoter. Such vectors have already been described to drive transgene expression in erythroid [1–3], T lymphocytic [4, 5], macrophagic [6], or megakaryocytic (MK) lineages [7, 8].

MK lineage is an attractive target for gene transfer. First, MK precursors are large polyploid cells with a high biosynthetic capacity, all alleles being transcriptionally active [9], potentially permitting a high level of transgenic protein expression. Along differentiation, MK progenitors synthesize specific platelet proteins that are considered differentiation markers [10], among which are platelet factor 4 and the glycoprotein (Gp) IIb/IIIa and GpIb-V-IX complexes [11]. Second, the final step of differentiation involves transendothelial cytoplasmic fragmentation to produce platelets [12] that play an essential role in hemostasis. Although megakaryocytes are rare and only represent 0.5% of the bone marrow (BM) cell population, they individually shed 10³ platelets, and approximately 10¹¹ platelets are produced daily in humans.

The importance of limiting transgene expression to the MK lineage is obvious in gene therapy approaches aiming at correcting the expression of proteins essential to platelet functions, as it is the case in Bernard-Soulier and Glanzman syndromes and in Von Willebrand disease [7, 8, 13]. Moreover, targeting the expression of the transgene to the MK lineage has also been used in a gene therapy strategy for hemophilia A [14, 15].

Because of the paucity of MKs in the BM and the absence of de novo transcription in platelets, most studies of MK gene regulation have been done in multipotential hematopoietic cell lines that retain some capacity to differentiate into MK cells. Among the MK promoters, one of the most studied is the GpIIb promoter, which was thought to induce specific MK expression by being switched off in non-MK cells [16]. However, GpIIb promoter-driven expression was detected in hematopoietic immature progenitors in transgenic models [17, 18] and in avian multilineage hematopoietic progenitor cells [19]. In agreement with these data, it has recently been shown that the GpIIb/GpIIIa protein complex was expressed at the surface of mouse and human immature hematopoietic progenitors [20, 21].

Therefore, the GpIIb promoter was expected to be active, when transduced using a lentiviral vector, in immature hematopoietic cells not yet engaged in MK differentiation. We thus looked for a promoter that would drive transgene expression later during MK differentiation and elected the GpIb promoter. In this study, we examined, in the context of the progeny of lentivirally transduced immature CD34⁺ human hematopoietic cells, the transcriptional activity of a 322-base-pair (bp) fragment of the GpIb promoter, already described to be specifically active in human MK cell lines after transfection [22].

We first replaced the constitutive EF1 promoter sequence of the human immunodeficiency virus (HIV)-derived SIN lentiviral vector TRIPU3-EF1-GFP [23] by short promoter sequences of the human GpIb (–322/+19 bp) [22] or GpIIb (–813/+33 bp) [24] genes to drive the expression of the green fluorescent protein (GFP) coding sequence. We then examined GFP expression in the progeny of transduced human CD34⁺ cells allowed to differentiate in vitro along different hematopoietic lineages. Transduced CD34⁺ cells were also injected into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, and GFP expression was analyzed either directly in the BM or after in vitro culture of CD34⁺ human cells sorted from the BM of transplanted mice. Analyses of GFP expression in transduced cells were performed together with analyses of differentiation-marker expression by flow cytometry and immunoelectron microscopy. Cells transduced with the EF1-GFP vector were used as control in all experiments. We especially compared the expression of GFP driven either by the GpIb or GpIIb promoters in early progenitors and along the differentiation toward various hematopoietic cell lineages of cord blood (CB), BM, or peripheral-blood mobilized (PBM) CD34⁺ cells. In the present study we demonstrate that, in contrast to the GpIIb promoter, the minimal human GpIb promoter (–322/+19 bp) tightly restricts transgene expression to the MK lineage after transduction of human hematopoietic immature cells.

	MATERIALS AND METHODS

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

 
Lentiviral Constructs
A fragment of the human GpIb promoter was amplified by polymerase chain reaction (PCR) on normal human DNA using forward and reverse primers in which MluI and BamHI restriction sites (italicized in the sequence) were inserted. The forward primer (5'-CAGCCTCCCAAACGCGTTCTGGGATTACAG-3') matched (bold) nucleotides 2,473–2,484 and 2,486–2,497, and the reverse primer (5'-AGGCAGGCAGAAAGGATCCTCCGAAGGCACAG-3') matched (bold) nucleotides 2,800–2,814 and 2,816–2,829 of the GpIb gene (GenBank M22403 [GenBank] ).

The human GpIIb promoter sequence extending from –813 to +32 bp was generated by PCR amplification from the pBLCAT3 plasmid containing the GpIIb promoter (GenBank M22568 [GenBank] ) [24] (kind gift from P. Albanese). The following primers, containing restriction sites italicized in the sequences, were used: forward primer (Mlu1 site) 5'-TTAATGGTTAAACGCGTGAGCTCCATCACAG-3' and reverse primer (BamH1 site) 5'-AGATCTGGATCCGGATCCTCTAGTCTTCCT-3'.

The PCR products were cloned into pCR2.1-TOPO plasmid (Invitrogen, SARL, Cergy-Pontoise, France, http://www.invitrogen.com) and one clone of each promoter fragment, the sequence of which has been checked on both strands, was selected. The Mlu1 and BamH1 restriction fragments of these plasmids were used to exchange the EF1 promoter of the TRIPU3-EF1-GFP vector with the GpIb and the GpIIb promoter sequences to obtain the TRIPU3-GpIb-GFP and the TRIPU3-GpIIb-GFP vectors, respectively.

Vector Production
Vector particles were produced by transient cotransfection of 293 T cells with a lentiviral vector plasmid, an encapsidation plasmid lacking all accessory HIV-1 proteins, and a G protein of the vesicular somatitis virus (VSV-G) envelope expression-plasmid, as previously described [23]. Viral titers, measured on the MK DAMI cell line [25], were 9.5, 9.3, and 7.3 10⁸/ml for EF1-GFP, GpIb-GFP, and GpIIb-GFP vectors, respectively.

Selection of CD34⁺ Cells
CB samples, BM, and PBM cells were collected with the informed consent of the mothers or the patients, according to approved French institutional guidelines. We isolated CD34⁺ cells by immunomagnetic selection (Miltenyi Biotec, Paris, http://www.miltenyibiotec.com) as recommended by the manufacturer, and purified CD34⁺ cells were immediately used in transduction experiments or frozen for subsequent usage.

Transduction of CD34⁺ Cells
Human CD34⁺ cells were plated at 10⁶ cells per milliliter in serum-free medium (Iscove's modified Dulbecco's medium [Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com] supplemented with 15% BIT 9500 [Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com]) in the presence of recombinant human (rhu) stem cell factor (SCF; 100 ng/ml; Amgen, Thousand Oaks, CA, http://www.amgen.com), rhu-Flt3-ligand (FL; 100 ng/ml; Immunex, Seattle, http://immunex.com), rhu-interleukin 3 (IL-3; 60 ng/ml; Novartis International, Basel, Switzerland, http://www.novartis.com), and rhu-thrombopoietin (TPO; 10 ng/ml; PromoCell, Heidelberg, Germany, http://www.promocell.com) together with lentiviral vector particles for an overall period of 72 hours, as previously described [26]. Cells were then washed and analyzed by flow cytometry for CD34 and GFP expression or used for hematopoietic cell cultures or for injection into NOD-SCID mice. In these experiments, we used transduction conditions to obtain a low copy number of the vector integrated in the majority of the cells.

Hematopoietic Cell Cultures and Flow Cytometry Analysis
Erythrocytic cultures were performed in serum-free culture conditions supplemented with IL-3 (10 ng/ml), IL-6 (10 ng/ml), and SCF (25 ng/ml). Cells were initially plated at 1.5 x 10⁵ per milliliter. After 6 days, erythropoietin was added at a final concentration of 2 IU/ml for 4 additional days. Cells were analyzed at day 13 of overall culture.

Maturing MK cells were observed 6–8 days after plating 5 x 10⁴ transduced cells per milliliter in serum-free culture medium in the presence of TPO (20 ng/ml) and SCF (5 ng/ml). Cells were analyzed at day 13 of overall culture in Figure 1A and 1D. For the comparison between GpIb and GpIIb promoters, analyses were performed every 2 or 3 days up to day 22 of the overall culture.

Granulocytes were allowed to differentiate in serum-free culture conditions in the presence of IL-3 (10 ng/ml), FL (50 ng/ml), SCF (10 ng/ml), and granulocyte colony-stimulating factor (CSF) (15 ng/ml). Cells were initially plated at 1 x 10⁵ per milliliter, and fresh medium was added at day 7. Cells were analyzed at days 13 and 17 of overall culture.

For monocytic cell cultures, cells were plated at 2 x 10⁵ per milliliter in serum-free culture conditions supplemented with FL (25 ng/ml) and macrophage CSF (25 ng/ml). At day 6, IL-3 was added (10 ng/ml), and cultures were prolonged for 4–5 days. Cells were analyzed at day 14 of overall culture.

Lymphocyte B/natural killer cells were obtained after 3 weeks of culture of transduced cells in 24-well plates (Falcon; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) on a previously established confluent layer of MS-5 cells in RPMI 1640 (Gibco-BRL) with 10% human AB serum (Institut Jacques Boy, Reims, France, http://www.biotechjboy.com), 5% pretested fetal calf serum (HCC-6400; Stem Cell Technologies), SCF (50 ng/ml), rhu IL-2 (1 ng/ml), and rhu IL-15 (1 ng/ml). Cells were initially seeded at 1 x 10⁴ per milliliter.

Dendritic cell culture was performed in serum-free culture conditions in the presence of tumor necrosis factor- (2.5 ng/ml), IL-4 (2 ng/ml), granulocyte macrophage CSF (10 ng/ml), and SCF (10 ng/ml). Cells were examined by fluorescence-activated cell sorting analysis at day 10 of overall culture.

Flow cytometry analyses were performed using fluorochrome-coupled monoclonal antibodies (MoAbs) directed against CD14 (clone IM0650 and IM2640) and CD15 (IM1423) for granulocytic-macrophagic differentiation, CD19 (A07771) for B lymphocytes, CD34 (IM2472) for progenitor cells, CD41a (A07781 [GenBank] ) and CD42b (IM1417) for MK differentiation, CD56 (NKH1) for natural killer (NK) lymphocytes, and glycophorin A (GPA) (KC16) for erythroid cells. All of the MoAbs were from Immunotech (Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp) and were coupled with phycoerythrin (PE), PE-cyanin 5 (PC5), or allophycocyanin (APC) fluorochromes. In all flow cytometry analyses, nonspecific staining was checked using corresponding isotypic controls (IgG1, IgG2a, and IgM) coupled to the same fluorochrome.

Semisolid Cloning Assays
Transduced CD34⁺ cells were plated in duplicate in methylcellulose under previously described conditions [27], and colony-forming cells (CFC) were counted at day 12 and analyzed by fluorescence microscopy. Transduced CD34⁺ cells were also plated in semisolid serum-free medium using the fibrin clot technique (MegaCult-C; Stem Cell Technologies). Number and fluorescence of colony-forming unit (CFU)-MK were examined at day 12.

Transplantation into NOD-SCID Mice
NOD-SCID mice were produced in the animal facilities of the Institut National de la Santé et de la Recherche Médicale U602 (Paul Brousse Hospital, Villejuif, France). Cells expanded from 10⁵ CD34⁺ cells during the 72 hours of the transduction protocol were washed and injected into sublethally irradiated (2.5 Gy) NOD-SCID mice. Twelve weeks after transplantation, BM cells from grafted animals were analyzed by flow cytometry for the presence of human CD45⁺ cells (PE-coupled MoAb against CD45 [IM0782] from Beckman Coulter [Fullerton, CA, http://www.beckmancoulter.com]). Mice were considered to be positive for human cell engraftment when at least 0.1% of CD45⁺ cells were detected among mouse BM cells. BM cells of positive mice were then phenotyped by flow cytometry analysis for expression of GFP together with expression of lineage differentiation markers as described above. Human CD34⁺ cells from the most engrafted mice were isolated by immunomagnetic selection from pooled BM cells and seeded in the various hematopoietic cell culture conditions, as described above. We obtained large amounts of differentiated cells in erythrocytic, granulocytic-macrophagic, B lymphocytic, and NK cell lineages in their respective culture conditions (Fig. 1D), whereas standard MK culture conditions were poorly efficient to obtain MK expansion in a reproducible manner. This is probably related to the fact that human hematopoiesis in NOD-SCID mice is mostly oriented toward B lymphocyte differentiation. Indeed, MK progenitors are very rare within the human CD34+ cells present in the mouse BM, which, in addition to the fact that maturing MK cells undergo endomitosis rather than mitosis, most likely explains the poor expansion efficiency in culture.

Immunogold Electron Microscopy
Mouse BM was removed by flushing femurs, and cells were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer pH 7.4, then embedded in sucrose and frozen in liquid nitrogen. Cryosections were made using an ultracryomicrotome (Reichert Ultracut S., Leica, Wetzler, Austria, http://www.leica-microsystems.com), and ultrathin sections mounted on formvar-coated nickel grids were prepared. Briefly, the sections were incubated for 15 minutes with phosphate-buffered saline (PBS) 15% glycine; 5 minutes with PBS 15% glycine, 0.1% bovine serum albumin (BSA); and 20 minutes with PBS 15% glycine, 0.1% BSA, 10% normal goat serum followed by 2 hours of incubation with monoclonal anti-GFP antibody (Clontech, Palo Alto, CA, http://www.clontech.com). The primary antibodies were diluted 1:100 in PBS 15% glycine, 0.1% BSA, 4% normal goat serum. After extensive rinsing in PBS 15% glycine, 0.1% BSA, sections were incubated for 1 hour with gold-labeled secondary goat anti-rabbit antibody with a gold particle with a size of 10 nm (GAR 10; British Biocell, Cardiff, U.K., http://www.british-biocell.co.uk). Sections were then washed for 30 minutes with PBS 15% glycine, stained with 4% uranyl acetate in 2% methylcellulose for 10 minutes, and air-dried. Examination was performed with a Philips CM10 electron microscope [28].

	RESULTS

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

 
GpIb Promoter Drives MK-Specific Expression in Human CD34⁺ Cells Differentiated Both In Vitro and In Vivo
CD34⁺ human CB cells transduced with either EF1-GFP or GpIb-GFP vectors were seeded in various culture conditions, allowing differentiation into granulocytes, monocytes, erythrocytes, MK, dendritic cells, and B or NK lymphocytes. Whereas the transduction efficiency and the proportion of differentiated cells of each lineage were slightly different in the five independent experiments performed, within each experiment the percentage of differentiated cells was identical among cells transduced with either of the two vectors. Importantly, the GpIb and EF1 promoters allowed GFP expression in comparable proportions of differentiating MK cells expressing both CD41a (GpIIb) and CD42b (GpIb) surface markers, whereas GFP expression driven by the two promoters was strikingly different in all other lineages studied. From the same pool of CD34⁺ cells transduced with either of the two vectors, EF1 sequence promoted ubiquitous expression of GFP in differentiated erythroid, granulocytic-macrophagic, B and NK lymphoid, and dendritic cells in proportions comparable with those observed in differentiating MKs. In contrast, the GpIb promoter did not allow expression of GFP in GPA⁺ erythroid cells, CD15⁺ and/or CD14⁺ granulocytic-macrophagic cells, CD11c dendritic cells, or CD19⁺ B and CD56⁺ NK lymphocytes (Fig. 1A).

View larger version (65K): [in this window] [in a new window]   Figure 1. MK-specific expression of GFP under GpIb promoter transcriptional control during differentiation of transduced CD34+ human cord blood (CB) cells. Representative flow cytometry analyses of the progeny of CB CD34+ transduced cells performed with the indicated PE-, APC-, or PC5-conjugated antibodies. Lineage-specific labelings are shown in correlation with GFP fluorescence. (A): Analysis of cells derived in vitro in different culture conditions from the same batch of CD34+ CB cells transduced with either GpIb-GFP or EF1-GFP vectors. One representative out of five experiments. (B): Flow cytometry analysis of total mouse bone marrow (BM) cells of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice 12 weeks after transplantation with transduced human CB CD34+ cells after labeling with the indicated human-specific fluorochrome-conjugated antibodies. Data are from one representative out of four and five mice grafted with cells transduced with either GpIb-GFP or EF1-GFP vectors, respectively. (C): Flow cytometry analysis of platelet-rich plasma (PRP) from blood of one representative out of five NOD-SCID mice 12 weeks after transplantation with GpIb-GFP transduced human CB CD34+ cells. Few CD41a+/CD42b+ human platelets, among which some are GFP+, are detected (right panels). Left panels represent the same analysis on the PRP of a nongrafted mouse. (D): CD34+ human cells were sorted from the BM of engrafted mice, placed under various hematopoietic culture conditions, and analyzed for specific hematopoietic lineage markers and GFP expression. Abbreviations: APC, allophycocyanin; FSC, forward scatter; GFP, green fluorescent protein; GpIb, glycoprotein Ib; PC5, phycoerythrin-cyanin 5; PE, phycoerythrin; SSC, side scatter.

We also examined expression of GFP in the in vivo-derived progeny of transduced CB CD34⁺ cells in the NOD-SCID model of xenogenic transplantation of human cells. NOD-SCID mice receiving CD34⁺ cells transduced with EF1-GFP (four mice injected) or GpIb-GFP (five mice injected) lentiviral vectors (supplemental online Fig. 1) were sacrificed 12 weeks after transplantation. Bone marrow cells were analyzed for CD45 human-specific hematopoietic marker expression. All mice were successfully engrafted with means of 12% (14%, 15%, 9%, 12%) and 25% (58%, 17%, 17%, 8%, 28%).

We first analyzed GFP expression in association with different lineage markers in human cells present in the BM (Fig. 1B). In the total human CD45⁺ cell population, EF1 promoter induced GFP expression in 76% of cells, versus only 4% for GpIb promoter. In B lymphocytes and granulocytes, EF1-GFP vector allowed 60%–80% of cells to express GFP. In contrast, cells that expressed GFP driven by the GpIb promoter were very rare, less than 5% in all cases. As usual, in this xenogenic model, erythroid and MK cells were barely detectable in the mouse BM using GPA and CD41a/CD42b expression, respectively [28]. Platelet-rich plasmas (PRP) from the same grafted mice were also analyzed by flow cytometry for CD41a, CD42b, and GFP expression. Although rare, human platelets were clearly detected, among which some were GFP⁺ (Fig. 1C). We sorted human CD34⁺ cells from the mouse BM that exhibited the highest proportion of human cell engraftment and cultured them in all the conditions described above. We examined GFP expression in differentiated cells and confirmed that, among the progeny of the NOD-SCID repopulating cell (SRC) transduced with the GpIb-GFP vector, none (including erythroid) but MK cells expressed GFP (Fig. 1D).

A second experiment performed in NOD-SCID mice (mice transplanted with CB CD34⁺ cells transduced with either the EF1-GFP or the GpIb-GFP vectors, three mice each) gave comparable results (not shown). These results obtained in the NOD-SCID model were confirmed by morphological identification, by immunoelectron microscopy, of human MKs as the only GFP-expressing cells in BM of mice grafted with GpIb-GFP transduced CD34⁺ cells (Fig. 2).

View larger version (101K): [in this window] [in a new window]   Figure 2. Electron-microscopy photograph from immunogold labeling for green fluorescent protein (GFP). Human megakaryocytes differentiated within nonobese diabetic-severe combined immunodeficient mouse bone marrow after transplantation of either CD34+ cells transduced with the GpIb-GFP vector (upper panel) or nontransduced CD34+ cells (lower panel). Arrows indicate GFP labeling in the cytoplasm (magnification 70,000). Abbreviation: N, cell nuclear membrane.

The GpIb Promoter Drives a More Restricted MK-Specific Expression than the GpIIb Promoter During the Initial Steps of CD34⁺ Cell MK Differentiation

CD34⁺ human cells sorted from the same samples were transduced with EF1-GFP, GpIb-GFP, or GpIIb-GFP vector and then placed under culture conditions allowing MK differentiation. We performed these experiments using CD34⁺ cells sorted from CB (n = 2), BM (n = 2), and PBM (n = 2). The GFP expression, together with the expression of three membrane markers (CD34 as immature hematopoietic cell marker, CD41a and CD42b as MK cell markers), was then analyzed daily for up to 3 weeks by flow cytometry. The kinetics of MK differentiation was the same no matter which vector was used to transduce the CD34⁺ cells in each of the eight experiments.

Figure 3A illustrates, in one representative experiment performed with CB cells transduced with either GpIb-GFP or GpIIb-GFP vectors, the differences observed in GFP expression at the very beginning of the MK culture. At day 4 of the overall culture, the cell population expressing GFP in the progeny of CD34⁺ cells represented 5% and 23%, and the CD34⁺GFP⁺ cell population represented 4.2% and 18% of the cells transduced with GpIb-GFP or GpIIb-GFP vectors, respectively (Fig. 3A, left panels, right and upper/right quadrants). Among these CD34⁺GFP⁺ cells, 47% expressed neither CD41a nor CD42b markers when transduced with the GpIb-GFP vector in contrast to 80% with the GpIIb-GFP vector (Fig. 3A, middle/left panels). This showed that transduction with the GpIb-GFP vector induced GFP expression in a lower proportion of immature cells not yet engaged in the MK differentiation, compared with the GpIIb-GFP vector. At the same time (day 4), within more mature CD34^– cells, the difference in the proportion of GFP⁺ cells that seemed not engaged in terminal MK differentiation (CD41a^–/CD42b^– cells) is not so important: 54% versus 65% of the CD34^–GFP⁺ cells when transduced with GpIb-GFP and GpIIb-GFP vectors, respectively. These CD34^–/CD41a^–/CD42b^– cells most probably corresponded in part to early maturing MK cells that did not yet express MK surface markers. However, we failed to evidence intracellular labeling with anti-CD41a and anti-CD42b antibodies by flow cytometry analysis after permeation of the cells. At day 8 of the overall culture (Fig. 3A, middle/right and right panels), the same observations were made, although the proportion of CD34⁺ cells was much lower, since most of the cells were already engaged in MK differentiation.

View larger version (40K): [in this window] [in a new window]   Figure 3. Expression of GFP under GpIb, GpIIb, and EF1 promoter transcriptional control during MK differentiation of transduced CD34+ cells from CB, BM, and PBM. Flow cytometry analysis after labeling with the indicated PE-, PECy5-, or APC-conjugated antibodies. Cells were examined for CD34 expression in correlation with GFP fluorescence or within the GFP+ cell population for CD41a and CD42b expression. (A): Results presented correspond to one representative out of four experiments. Right and left panels present results obtained with GpIb-GFP- and GpIIb-GFP-transduced CB cells, respectively. (B): Histograms represent the evolution of respective proportions of two cell populations among the MK differentiation cultures of CB, BM, and PBM CD34+ cells transduced with each of the three vectors: CD41a–CD42b–GFP+ (black bars) and CD41a+CD42b+GFP+ (white bars) cell populations. Results presented correspond to one representative out of two kinetics analyses for each cell population. Abbreviations: APC, allophycocyanin; BM, bone marrow; CB, cord blood; GFP, green fluorescent protein; GpIb, glycoprotein Ib; GpIIb, glycoprotein IIb; PBM, peripheral-blood mobilized; PECy5, phycoerythrin-cyanin 5; PE, phycoerythrin.

It should be noted that the level of GFP within individual cells, in terms of mean of fluorescence intensity, was always lower in the progeny of CD34⁺ cells transduced with GpIb-GFP vector compared with cells transduced with GpIIb-GFP vectors (supplemental online Fig. 2). This smaller quantity of GFP per cell could result from the fact that, being expressed later in GpIb-GFP-transduced cells, GFP accumulated less than in cells transduced with the other vectors. But it could also be explained by a weaker promoter activity of the GpIb promoter fragment compared with the GpIIb promoter. Of course, the two explanations are not mutually exclusive.

	DISCUSSION

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

 
Our data indicate that the GpIb promoter activity is more restricted to differentiating MK cells than the GpIIb promoter. Results obtained with the GpIIb promoter were anticipated, since GpIIb protein is expressed by undifferentiated progenitors [17, 18] and by the bipotent erythro-MK progenitor [20, 21], whereas GpIb protein has been detected only later during MK differentiation, within the GpIb-V-IX glycoprotein membrane complex. However, this did not predict that the GpIb promoter would prove to be so specific. Indeed, the detection of endogenous protein complexes at the cell membrane does not always strictly parallel the transcriptional activity of the individual corresponding genes. Indeed, mRNA can be expressed much earlier in the differentiation process than the protein it encodes, this being especially true when post-translational modifications and multiple protein assembly are necessary for the processing of transmembranous complexes such as the GpIb-V-IX. This point probably explains GFP expression in part of the cells that do not still express CD41a or CD42b surface markers. Another source of potential discrepancy between transgene versus endogenous protein expression is that promoter fragments may not contain all the regulatory sequences of the endogenous genes. It is quite remarkable that the short GpIb regulatory sequence that we used exhibited such a tight MK-specific regulation.

Considering these arguments, we do not draw any conclusion of our study concerning the regulation of endogenous promoters. But, in this study, we clearly demonstrated an MK-specific activity of the 238-bp GpIb promoter in the context of a lentiviral vector, allowing the tightly restricted expression of the transgene in the MK lineage after transduction of CD34⁺ human cells in vitro and in vivo in the xenogenic NOD-SCID mouse model. We also demonstrated that, in comparison, the GpIIb promoter is not as efficient as the GpIb promoter to strictly restrict the transgene expression to the MK lineage in the progeny of lentivirally transduced CD34⁺ cells, since it allowed transgene expression in a significant proportion of immature CD34+ cells. The very short 238-bp GpIb promoter is thus a promising tool to strictly restrict transgene expression to MK and platelets, in the context of a lentiviral vector used for fundamental studies or for a gene therapy strategy targeting hematopoietic stem cells [8, 13, 15].

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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

 
B.I. and F.L. contributed equally to this article. We thank S. Gisselbrecht, P.H. Roméo, and F. Pflumio for helpful and stimulating discussions. We acknowledge F. Pflumio for advice and help with the mouse experiments. We thank A. Schmitt for his expertise in electron-microscopy experiments. We wish to thank particularly the midwives and Dr. Y. Rouquet from the Clinique des Noriets, Vitry-sur-Seine, for providing the numerous cord blood samples, and Drs. C. Le Bousse-Kerdiles and J.J. Lataillade for providing bone marrow samples. This project was supported in part by grants from INSERM, Association Française contre les Myopathies (AFM), and ANR-GIS Maladies rares. C.L.-B. was recipient of a fellowship from the AFM. F.L. is currently affiliated with Service d'Hématologie, Hôpital Minjoz, Université de Franche-Comté, Besançon, France.

	REFERENCES

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

 

1. Pawliuk R, Westerman KA, Fabry ME et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001;294:2368–2371.[Abstract/Free Full Text]

2. May C, Rivella S, Callegari J et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000;406:82–86.[CrossRef][Medline]

3. Moreau-Gaudry F, Xia P, Jiang G et al. High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors. Blood 2001;98:2664–2672.[Abstract/Free Full Text]

4. Indraccolo S, Minuzzo S, Roccaforte F et al. Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors. Blood 2001;98:3607–3617.[Abstract/Free Full Text]

5. Moreau T, Bardin F, Imbert J et al. Restriction of transgene expression to the B-lymphoid progeny of human lentivirally transduced CD34+ cells. Mol Ther 2004;10:45–56.[CrossRef][Medline]

6. Gough PJ, Raines EW. Gene therapy of apolipoprotein E-deficient mice using a novel macrophage-specific retroviral vector. Blood 2003;101:485–491.[Abstract/Free Full Text]

7. Shi Q, Wilcox DA, Morateck PA et al. Targeting platelet GPIbalpha transgene expression to human megakaryocytes and forming a complete complex with endogenous GPIbbeta and GPIX. J Thromb Haemost 2004;2:1989–1997.[CrossRef][Medline]

8. Fang J, Hodivala-Dilke K, Johnson BD et al. Therapeutic expression of the platelet-specific integrin, alphaIIbbeta3, in a murine model for Glanzmann thrombasthenia. Blood 2005;106:2671–2679.[Abstract/Free Full Text]

9. Raslova H, Roy L, Vourc'h C et al. Megakaryocyte polyploidization is associated with a functional gene amplification. Blood 2003;101:541–544.[Abstract/Free Full Text]

10. Rabellino EM, Levene RB, Leung LL et al. Human megakaryocytes. II. Expression of platelet proteins in early marrow megakaryocytes. J Exp Med 1981;154:88–100.[Abstract/Free Full Text]

11. Breton-Gorius J, Vainchenker W. Expression of platelet proteins during the in vitro and in vivo differentiation of megakaryocytes and morphological aspects of their maturation. Semin Hematol 1986;23:43–67.[Medline]

12. Long MW. Megakaryocyte differentiation events. Semin Hematol 1998;35:192–199.[Medline]

13. Pergolizzi RG, Jin G, Chan D et al. Correction of a murine model of von Willebrand disease by gene transfer. Blood 2006;108:862–869.[Abstract/Free Full Text]

14. Yarovoi HV, Kufrin D, Eslin DE et al. Factor VIII ectopically expressed in platelets: Efficacy in hemophilia A treatment. Blood 2003;102:4006–4013.[Abstract/Free Full Text]

15. Shi Q, Wilcox DA, Fahs SA et al. Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies. J Clin Invest 2006;116:1974–1982.[CrossRef][Medline]

16. Prandini MH, Martin F, Thevenon D et al. The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements. Blood 1996;88:2062–2070.[Abstract/Free Full Text]

17. Tropel P, Roullot V, Vernet M et al. A 2.7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells. Blood 1997;90:2995–3004.[Abstract/Free Full Text]

18. Tronik-Le Roux D, Roullot V, Poujol C et al. Thrombasthenic mice generated by replacement of the integrin alpha(IIb) gene: Demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment. Blood 2000;96:1399–1408.[Abstract/Free Full Text]

19. Ody C, Vaigot P, Quere P et al. Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells. Blood 1999;93:2898–2906.[Abstract/Free Full Text]

20. Debili N, Robin C, Schiavon V et al. Different expression of CD41 on human lymphoid and myeloid progenitors from adults and neonates. Blood 2001;97:2023–2030.[Abstract/Free Full Text]

21. Mitjavila-Garcia MT, Cailleret M, Godin I et al. Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells. Development 2002;129:2003–2013.[Medline]

22. Hashimoto Y, Ware J. Identification of essential GATA and Ets binding motifs within the promoter of the platelet glycoprotein Ib alpha gene. J Biol Chem 1995;270:24532–24539.[Abstract/Free Full Text]

23. Sirven A, Ravet E, Charneau P et al. Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors. Mol Ther 2001;3:438–448.[CrossRef][Medline]

24. Prandini MH, Uzan G, Martin F et al. Characterization of a specific erythromegakaryocytic enhancer within the glycoprotein IIb promoter. J Biol Chem 1992;267:10370–10374.[Abstract/Free Full Text]

25. Greenberg SM, Rosenthal DS, Greeley TA et al. Characterization of a new megakaryocytic cell line: The Dami cell. Blood 1988;72:1968–1977.[Abstract/Free Full Text]

26. Amsellem S, Ravet E, Fichelson S et al. Maximal lentivirus-mediated gene transfer and sustained transgene expression in human hematopoietic primitive cells and their progeny. Mol Ther 2002;6:673–677.[CrossRef][Medline]

27. Issaad C, Croisille L, Katz A et al. A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays. Blood 1993;81:2916–2924.[Abstract/Free Full Text]

28. Bruno S, Gunetti M, Gammaitoni L et al. Fast but durable megakaryocyte repopulation and platelet production in NOD/SCID mice transplanted with ex-vivo expanded human cord blood CD34+ cells. STEM CELLS 2004;22:135–143.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Nishikii, K. Eto, N. Tamura, K. Hattori, B. Heissig, T. Kanaji, A. Sawaguchi, S. Goto, J. Ware, and H. Nakauchi Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells J. Exp. Med., August 4, 2008; 205(8): 1917 - 1927. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 2006-0321v1 25/6/1571    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lavenu-Bombled, C. Articles by Dubart-Kupperschmitt, A. Search for Related Content PubMed PubMed Citation Articles by Lavenu-Bombled, C. Articles by Dubart-Kupperschmitt, A.