CD34.SUP.+.CD41.SUP.DIM .megakaryocytes progenitors and uses thereof for producing proplatelet-bearing MKs and/or platelets

Abstract

The invention relates to a method of producing CD34+CD4.sup.dim megakaryocyte (MK) progenitor cells, and substantially pure cell population of megakaryocyte precursor cells obtained by said method. The invention also relates to a method of producing proplatelet-bearing MKs and/or platelets using the CD34+CD4.sup.dim cells.

Claims

1. An ex vivo method of producing proplatelet-bearing megakaryocytes (MKs) and/or platelets comprising: a) culturing an isolated CD34.sup.+CD9.sup.−CD41.sup.+ cell population of MK progenitors in a serum-free culture medium comprising thrombopoietin (TPO), in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising proplatelet-bearing MKs and/or platelets; and b) collecting said cell population comprising proplatelet-bearing MKs and/or platelets.

2. The method according to claim 1, wherein in a), culturing is conducted for 5 to 9 days.

3. The method according to claim 1, wherein the serum-free culture medium comprises 20-100 ng/ml TPO.

4. The method according to claim 1, wherein said method comprises, prior to a): a0) culturing haematopoietic stem cells (HSC) in a serum-free culture medium comprising low-density lipoprotein (LDL), stem cell factor (SCF), TPO, IL-6 and IL-9, in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising CD34.sup.+CD9.sup.−CD41.sup.+ cells; and a1) isolating said CD34.sup.+CD9.sup.−CD41.sup.+ cells from said cell population.

5. The method according to claim 4, wherein the serum-free culture medium of a0) comprises 10-30 μg/ml LDL, 25-100 ng/ml SCF, 40-50 ng/ml TPO, 20-30 ng/ml IL-6 and 20-30 ng/ml IL-9.

6. The method according to claim 4, wherein in a0), culturing is conducted for 6 to 8 days.

7. The method according to claim 1, wherein in a) and a0), independently, the AhR antagonist is a compound of formula (I) ##STR00003## L is selected from the group consisting of —NR.sub.5a(CH.sub.2).sub.2-3-, —NR.sub.5a(CH.sub.2).sub.2NR.sub.5b—, —NR.sub.5a(CH.sub.2).sub.2S—, —NR.sub.5aCH.sub.2CH(OH)— and —NR.sub.5aCH(CH.sub.3)CH.sub.2—; wherein R.sub.5a and R.sub.5b are independently hydrogen or C.sub.1-4alkyl; R.sub.1 is selected from the group consisting of thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl of R.sub.1 is optionally substituted by 1 to 3 radicals independently selected from the group consisting of halo, cyano, C.sub.1-4alkyl, halo-substituted-C.sub.1-4alkyl, C.sub.1-4alkoxy, —S(O).sub.0-2R.sub.8a, and —C(O)OR.sub.8a, wherein R.sub.8a is hydrogen or C.sub.1-4alkyl; R.sub.2 is selected from the group consisting of —S(O).sub.2NR.sub.6aR.sub.6b, —NR.sub.6aC(O)NR.sub.6bR.sub.6c, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2-oxoimidazolidinyl, pyrazolyl, and indazolyl; wherein R.sub.6a, R.sub.6b and R.sub.6c are independently hydrogen or C.sub.1-4alkyl; and said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, or indazolyl of R.sub.2 is optionally substituted with 1 to 3 radicals independently selected from the group consisting of hydroxy, halo, methyl, methoxy, amino, —O(CH.sub.2).sub.nNR.sub.7aR.sub.7b, —OS(O).sub.2NR.sub.7aR.sub.7b and —NR.sub.7aS(O).sub.2R.sub.7b; wherein R.sub.7a and R.sub.7b are independently hydrogen or C.sub.1-4alkyl; R.sub.3 is selected from the group consisting of hydrogen, C.sub.1-4alkyl and biphenyl; and R.sub.4 is selected from the group consisting of C.sub.1-10alkyl, C.sub.1-4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said C.sub.1-10alkyl, C.sub.1-4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl of R.sub.4 can be optionally substituted with 1 to 3 radicals independently selected from the group consisting of hydroxy, C.sub.1-4alkyl and halo-substituted-C.sub.1-4alkyl.

8. The method according to claim 1, wherein in a), the AhR antagonist is StemRegenin 1 (SR1).

9. The method according to claim 1, wherein a) is performed by co-culture with hMSCs.

10. The method according to claim 7 wherein said hMSCs are obtained by a method comprising: i) isolating bone marrow mononuclear cells (BM-MNCs) from a human subject by Ficoll density gradient; ii) seeding isolated BM-MNCs in culture medium comprising 5-15% fetal bovine serum and 0.5-5 ng/mL fibroblast growth factor 2 (FGF-2); iii) culturing seeded cells for two days, and the discarding nonadherent cells and seeding collected adherent cells; iv) culturing adherent cells in culture medium comprising 10% fetal bovine serum and 0.5-5 ng/mL FGF-2, with replacement of culture medium twice a week with fresh culture medium until confluence; and v) harvesting hMSCs, seeding and culturing harvested cells until confluence in culture medium comprising 10% fetal bovine serum and 0.5-5 ng/mL FGF-2.

11. The method according to claim 1, which further comprises selecting CD41/CD61+ and CD42c+ cells from the collected cell population comprising proplatelet-bearing MKs and/or platelets.

12. The method according to claim 1, which further comprises washing the proplatelet-bearing megakaryocytes (MKs) and/or platelets and suspending the washed cells in an infusion buffer.

13. A method of producing megakaryocyte (MK) progenitor cells comprising: a0) culturing haematopoietic stem cells (HSC) in a serum-free culture medium comprising low-density lipoprotein (LDL), stem cell factor (SCF), TPO, IL-6 and IL-9, in presence of an aryl hydrocarbon receptor (AhR) antagonist or by co-culture with human mesenchymal stromal cells (hMSCs), for a time sufficient to obtain a cell population comprising CD34.sup.+CD9.sup.−CD41.sup.+ cells; and a1) isolating said CD34.sup.+CD9.sup.−CD41.sup.+ cells from said cell population.

14. The method according to claim 4, wherein in a) and/or a0), the AhR antagonist is StemRegenin 1 (SR1).

15. The method according to claim 4, wherein a) and/or a0) is performed by co-culture with hMSCs.

Description

FIGURES

(1) FIG. 1: Preservation of CD34 expression in MKs cultured in the presence of SR1. (A) MK differentiation protocol. Peripheral blood CD34.sup.+ cells were cultured in the absence (Ctrl) or presence of SR1 (1 μM) in a serum-free medium containing CC220 cytokine cocktail from day 0 to day 7 and with TPO (30 ng/mL) from day 7 to day 14. (B) Level of proliferation. Viable cells were counted on days 7 and 10 of culture using an automatic cell counter and the fold increase over the input of CD34.sup.+ cells on day 0 was calculated. (C) Proportion of CD34.sup.+ cells. The proportion of CD34.sup.+ cells was determined on the indicated days by flow cytometry after labeling with an R-PE-Cy5-conjugated anti-CD34 mAb. Experiments were performed at least three times (mean±SEM; two-way ANOVA and a Bonferroni post-test, n.s. P>0.05, ***P<0.001).

(2) FIG. 2: Increased production of proplatelets and platelet-like elements in the presence of SR1. CD34.sup.+ cells were cultured as in FIG. 1A and analyses were performed on day 14. (A) Quantification of the percentage of MKs extending proplatelets (34.6±2.1% with SR1 versus 11.5±4.5% for the control; mean±SEM in 3 experiments; Student's t-test *P<0.05). (B) Release of platelets. The cell suspension was subjected to multiple pipetting and platelet-like elements were detected and counted by flow cytometry. Upper panel: Representative gating strategy based on the forward and side scattering properties and CD41/CD42 expression of the cells. Lower panel: Number of platelet-like elements per cell seeded on day 7 (7.92±3.25 for the control vs 20.72±5.19 with 1 μM SR1 vs 0.20±0.04 with 0.2 μM of the AhR agonist FICZ) mean±SEM in 3 to 5 experiments; two-way ANOVA and a Bonferroni post-test, n.s. P>0.05).

(3) FIG. 3: Emergence of a CD34.sup.+CD41.sup.dim population in the presence of SR1. CD34.sup.+ cells were cultured as in FIG. 1A and analyses were performed on days 7 and 10. (A) Evolution of CD34 and CD41 expression. Representative flow cytometric dot plots in the absence (Ctrl) or presence of SR1. On day 7, three main populations were observed which were CD34.sup.+CD41.sup.− (purple), CD34.sup.+CD41.sup.+ (red) and CD34.sup.−CD41.sup.+ (blue) and represented respectively 23.1±1.3%, 59.9±2.3% and 9.7±1.1% of the total population in the control versus 22.4±1.5%, 68.9±1.8% and 3.6±0.3% in the presence of SR1. On day 10, the two main populations were CD34.sup.−CD41.sup.+ and CD34.sup.+CD41.sup.+. CD34.sup.−CD41.sup.+ cells amounted to 51.6±4.9% of the cells in the control versus 26.7±4.5% in the presence of SR1. The CD34.sup.+CD41 population (red) was more abundant in SR1-treated cultures (55.1±4.9%) than under control conditions (32.1±0.7%). A CD34.sup.+CD41.sup.+ subpopulation (region R2) with an intermediate level of CD41 expression, defined as CD34.sup.+CD41.sup.dim, was predominant in SR1 cultures. (B) Proportion of CD34.sup.+CD41.sup.dim cells. Bar graph representing the percentage of CD34.sup.+CD41.sup.dim cells gated in R.sub.2, which amounted to 16.8±1.4% of the total cells in the control versus 36.8±1.9% after SR1 treatment (mean±SEM in 8 experiments; Student's t-test, ***P<0.001). (C) Dot plots of forward light scattering versus CD41 expression showing the CD34.sup.+CD41.sup.dim population (red) and the CD34.sup.−CD41.sup.+ population (blue) from an SR1-treated culture on day 10.

(4) FIG. 4 Ploidy distribution of the CD34+CD41dim and CD34−CD41+ cells from an SR1-treated culture on day 10 (Student's t-test, *P<0.05, **P<0.005).

(5) FIG. 5. High capacity of CD34.sup.+CD41.sup.dim cells to produce proplatelets and platelet-like elements. (A) CD34.sup.+ cells cultured for 10 days in the presence of SR1 as in FIG. 1A were sorted according to their CD34.sup.+CD41.sup.dim and CD34.sup.−CD41 expression using a FACS Aria II flow cytometer and then cultured for 7 days in a medium containing TPO with or without SR1 (5 μM). (B) Quantification of the percentage of MKs extending proplatelets 7 days after seeding CD34.sup.+CD41.sup.dim cells (91.0±2.4% with SR1 versus 10.0±6.6% for the control; mean±SEM in 5 experiments; Student's t-test, ***P<0.001). (C) Number of platelet-like elements 7 days after seeding CD34.sup.+CD41.sup.dim cells (52.1±8.8 with SR1 versus 7.7±0.8 for the control; mean±SEM in 5-8 experiments; Student's t-test, **P<0.005).

(6) FIG. 6. Co-culture of CD34.sup.+ cells with hMSCs promotes platelet production and the emergence of a CD34.sup.+CD41.sup.dim population. (A) CD34.sup.+ cells were cultured as in FIG. 1A in the absence (Ctrl) or presence of a monolayer of hMSCs for up to 14 days. (B) Level of proliferation. Viable cells were counted on days 7 and 10 of culture using an automatic cell counter and the fold increase over the input of CD34.sup.+ cells was calculated (mean±SEM in 3 experiments; Student's t-test, n.s. P>0.05). (C) Quantification of culture-derived platelets. The cell suspension was subjected to multiple pipetting on day 14 of culture and platelet-like elements were detected and counted by flow cytometry (mean±SEM in 3 experiments; Student's t-test, *P<0.05).

(7) FIG. 7. (A) Effect of FICZ on platelet production. CD34.sup.+ cells were co-cultured on MSCs as in FIG. 5A in the presence or absence of the AhR agonist FICZ (0.2 μM). On day 14, platelet-like elements were counted by flow cytometry (20.6±1.3 vs 4.5±1.9 per cell seeded on day 7, with or without FICZ, respectively; mean±SEM in 3 experiments; Student's t-test, *** P<0.001? (B) CYP1B1 expression. qPCR analysis of CYP1B1 mRNA on day 10 in MKs co-cultured or not with MSCs or SR1 and with or without FICZ. Data are the mean values±SEM of 3 experiments.

(8) FIG. 8. (A) Evolution of CD34 and CD41 expression. Representative flow cytometric dot plots of CD34 and CD41 expression in the cell suspension on day 10 revealing a CD34.sup.+CD41.sup.dim population in MSC co-cultures. (B) Proportion of CD34.sup.+CD41.sup.dim cells. Bar graph representing the proportion of cells in the CD34.sup.+CD41.sup.dim region (mean±SEM in 3 experiments; Student's t-test, *P<0.05). (C) Ploidy distribution of the CD34.sup.+CD41.sup.dim cells from an MSC co-culture on day 10.

(9) FIG. 9. Comparable properties of CD34.sup.+CD41.sup.dim cells obtained after treatment with MSCs or SR1. (A) CD34.sup.+ cells were cultured for 10 days as in FIG. 1A in the presence of a monolayer of MSCs. CD34.sup.+CD41.sup.dim cells were sorted on day 10 and cultured for a further 7 days with TPO, TPO+5 μM SR1, or TPO+MSC. Panel i: Quantification of the percentage of MKs extending proplatelets. Panel ii: Quantification of culture-derived platelets. (B) CD34.sup.+ cells were cultured for 10 days as in FIG. 1A in the presence of 5 μM SR1. CD34.sup.+CD41.sup.dim cells were sorted on day 10 and cultured for a further 7 days with TPO, TPO+5 μM SR1, or TPO+MSC. Panel i: Quantification of the percentage of MKs extending proplatelets. Panel ii: Quantification of culture-derived platelets. Mean±SEM in 3-4 experiments.

(10) FIG. 10. Density plot allowing visualizing the cell population CD34.sup.+CD41.sup.dim.

(11) FIG. 11. Visualisation of the CD41 population among the CD9 population. A) Representative flow cytometric dot plot of CD34 and CD9 expression in the cell suspension on day 10. The region in the right lower inlet represents the CD34.sup.+CD9.sup.− cell population that is gated in Example 2. B) Representative flow cytometric dot plot of CD41 and CD9 expression in the cell suspension on day 10. The region in the right lower inlet of A) representing the CD34.sup.+CD9.sup.− cell population corresponds in the lower inlet of the CD41/CD9 dot plot which is CD9.sup.−CD41.sup.dim. It can be concluded that the CD9.sup.− cell population is identical with the CD9.sup.−CD41.sup.dim cell population.

(12) FIG. 12. Visualisation of the CD34.sup.+CD9.sup.−CD41.sup.dim cell population in comparison to the CD34.sup.+CD41.sup.dim cell population A) Representative flow cytometric dot plot of CD34 and CD9 expression in the cell suspension on day 10. The right lower inlet represents the CD34.sup.+CD9.sup.− cell population that is gated in Example 2. B) Representative flow cytometric dot plot of FSC/CD41.sup.+ expression of the CD34.sup.+CD9.sup.− cell population. The inlet in B) represents the CD34.sup.+CD9.sup.−CD41.sup.+ cell population. CD9.sup.− deselects CD41.sub.high. The remaining CD41.sup.+ cells as gated by FSC/CD41.sup.+ expression are thus CD41.sup.dim. The CD34.sup.+CD9.sup.−CD41.sup.+ cell population can thus be called a CD34.sup.+CD9.sup.−CD41.sup.dim cell population. C) Transposition of the CD34.sup.+CD9.sup.−CD41.sup.dim population on the CD34/CD41 graph in the cell suspension on day 10 (as used for example in FIG. 3 for obtaining the CD34.sup.+CD41.sup.dim cell population). The circle in C) indicates the cell population CD34.sup.+CD9 CD41.sup.+ in said CD34/CD41 graph. Comparison with for example FIG. 3A (Day 10) demonstrates that said CD34.sup.+CD9.sup.−CD41. Cell population corresponds to the CD34.sup.+CD9 cell population as indicated in FIG. 3 with region R2.

EXAMPLE 1

(13) Materials and Methods

(14) Isolation of CD34.sup.+ Cells

(15) CD34.sup.+ cells were recovered from leukodepletion filters obtained from the Etablissement Français du Sang-Alsace by adapting a procedure described by Ivanovic et al (Transfusion. 2006; 46:118-125.). Briefly, after 15 min incubation with RosetteSep® Human Granulocyte Depletion Cocktail (StemCell Technologies, Vancouver, Canada), mononuclear cells were isolated by Histopaque®-1077 (Sigma-Aldrich) density gradient separation for 30 min at 400 g. CD34.sup.+ cells were then isolated by positive selection using an immunomagnetic cell sorting system (AutoMacs, Miltenyi, Bergisch Galdbach, Germany). A viability of 83.30±1.96% and a CD34.sup.+ purity of 82.80±2.25% were routinely obtained (n=6).

(16) MK Differentiation in Culture

(17) CD34.sup.+ cells were seeded in 48-well plates at a density of 4×10.sup.4 per mL in StemSpan SFEM medium supplemented with 20 ng/mL human LDL and CC220 (1×), a cocktail of cytokines containing SCF, TPO, IL-6 and IL-9 (all from Stemcell Technologies), with or without addition of 1 μM SR1 (Cellagen Technology, San Diego, Calif.) (FIG. 1A). On day 7, the cells were harvested, washed and seeded at 5×10.sup.4/mL in StemSpan SFEM medium containing 30 ng/mL TPO with or without 1 μM SR1 for an additional 7 days. The cultures were incubated at 37° C. under normoxic conditions and 5% CO.sub.2. On days 7 and 10 of culture, the cells were counted, their viability was measured by propidium iodide exclusion in an automatic cell counter (ADAM, Digital-Bio, Korea) and the expression of CD34, CD41 and CD42b was analyzed in a Gallios flow cytometer using Kaluza software (Beckman Coulter, Villepinte, France). In some experiments, SR1 was replaced by the AhR agonist FICZ (Enzo life sciences, Villeurbane, France) added at 0.2 μM.

(18) In a second protocol, CD34.sup.+ cells were cultured in the presence of mesenchymal stromal cells (MSCs) isolated from human bone marrow (Guilloton F et al., Blood. 2012; 119:2556-2567). MSCs were maintained in α-MEM medium supplemented with 10% fetal bovine serum (Invitrogen, Cergy Pontoise, France) and 2 ng/mL recombinant human (rh) FGF2 (Peprotech, Rocky Hill, N.J.). CD34.sup.+ cells were added to a confluent layer of MSCs at a density of 4×10.sup.4/mL in 48-well plates in StemSpan SFEM medium supplemented with 20 ng/mL human LDL and CC220. On day 7, the cells in suspension were harvested, washed and co-cultured at 5×10.sup.4/mL on a new layer of confluent MSCs in StemSpan SFEM medium containing 30 ng/mL TPO for an additional 7 days (FIG. 5A).

(19) Cell Sorting

(20) The cells recovered on day 10 were incubated with a mixture of Alexa-488-conjugated anti-CD41 (ALMA.17) and PE-Cy7-conjugated anti-CD34 mAbs (BD Biosciences) for 30 min at 4° C. They were then washed in PBS-EDTA and incubated for 30 min in PBS containing 7-AAD (1/50) to select viable cells. The morphologic and sorting gates were determined by FMO (fluorescence minus one) analysis and megakaryocytic precursors were sorted at 500 cells/s according to their CD34/CD41 expression using a FACS Aria II flow cytometer (Becton Dickinson, Mountain View, Calif.) equipped with a 50 μm nozzle and two argon lasers operating at 500 mW and tuned to 488 and 360 nm, respectively (Coherent Radiation, Palo Alto, Calif.). The sorted CD34.sup.+CD41.sup.dim and CD34 CD41.sup.+ cells were then counted and seeded at 4×10.sup.4/mL in 48-well plates in StemSpan medium containing TPO with or without SR1 for 7 days (FIG. 4A).

(21) Analysis of MK Maturation

(22) Surface Markers. Cells were analyzed by flow cytometry (Gallios, Beckman Coulter, France) after labeling with anti-CD34-PE-Cy7 (Beckman Coulter, Fullerton, Calif.), anti-CD41-Alexa-488 (ALMA.17), anti-CD42c-PE (RAM.1) and anti-CD42d-Alexa-647 (V.1) mAbs for 30 min at 4° C. The cells were then washed and resuspended in PBS containing 7-AAD (1/50). The acquired data were analyzed with Kaluza software.

(23) Ploidy. Cells were incubated for 2 h at 37° C. with 10 μg/mL Hoechst 33342 (Sigma-Aldrich, Saint Quentin Fallavier, France) and then stained with anti-CD34-PE-Cy7 and anti-CD41-PE mAbs. The washed cells were resuspended in PBS containing 7-AAD and the ploidy distribution in the CD41 population was determined by two-color flow cytometry (Fortessa, BD Biosciences, Rungis, France). The acquired data were analyzed with BD FACSDiva software (BD Biosciences).

(24) Ultrastructure. Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 2% sucrose and processed as described previously (Eckly A et al., Blood. 2014; 123:921-930). Ultrathin sections were examined under a Philips CM120 Biotwin transmission electron microscope (FEI, Heindhoven, The Netherlands) at 120 kV.

(25) Quantification of Proplatelet-Bearing MKs

(26) The percentage of MKs extending proplatelets was determined in the culture wells by phase-contrast microscopy. In each culture, at least 100 MKs were analyzed and images were acquired using a Zeiss Axio Vert.A1 microscope with a 20× objective (Marly-le-Roi, France).

(27) Determination of the Number of Platelets Produced Per Seeded Cell

(28) CD34.sup.+ cells cultured for 7 days in the presence of CC220 (FIGS. 1A and 5A) or CD34/CD41 sorted cells (FIGS. 4A and 6A) were seeded in a medium containing TPO. On day 7, 1 μM PGE.sub.1 and 0.02 U/mL apyrase were added to the culture medium and the cells were gently passed 5 times through a P1000 pipet tip. The resulting suspension (200 μL) was incubated with anti-CD41-Alexa-647 and anti-CD42c-Alexa-488 mAbs for 15 min at room temperature before analysis in a Gallios flow cytometer. CD41/CD42c double positive events, having the same scattering properties as washed blood platelets, were counted as platelet-like particles and the number of particles per seeded cell, at day 7 or 10 following experiments, was determined.

(29) RNA Extraction

(30) CD41/61 cells were obtained on day 7 or 10 of culture using the antibody ALMA.17 and magnetic beads (EasySep® “Do-It-Yourself” Selection Kit, StemCell Technologies). Total RNA were extracted using an RNeasy® Mini kit (QIAGEN) following manufacturer's instructions. Quantity and quality of total RNA for all samples were evaluated by measuring OD at 260 nm and concentration was adjusted at 50 ng/ml. The RNA samples were stored at −80° C. until use. qRT-PCR was applied under standard conditions using the SYBR Green Master Mix kit on the ABI Prism 7900 Sequence Detection System (PerkinElmer-Cetus, Courtaboeuf, France). The primers for genes were chosen with the assistance of the Oligo 6.0 program (National Biosciences, Plymouth, Minn.) and have been previously described (Bieche I. et al., Pharmacogenetics and genomics. 2007; 17:731-742).

(31) Statistics

(32) Statistical significance was determined by means of Student's t-test or two-way Anova followed by a Bonferroni post-test. Data were analyzed using Graphpad Prism 5 software.

(33) Results

(34) SR1 Sustains CD34 Expression in MKs Differentiated from Peripheral Blood CD34.sup.+ Cells

(35) We evaluated the effect of the AhR antagonist SR1 on the expansion of MK precursors. SR1 (50 μM) was added on days 0 and 7 in a two-step culture protocol where peripheral blood CD34.sup.+ cells (Peytour Y et al., Transfusion. 2010; 50:2152-2157) were first expanded for 7 days in the presence of CC220, an optimized mix of SCF, TPO, IL-6 and IL-9, and then differentiated for a further 7 days in the presence of TPO alone (FIG. 1A).

(36) Using this protocol more than 90% of the control cells, cultured without SR1, were double positive for the platelet markers CD41 and CD42 on day 12 and displayed the hallmark features of fully mature MKs in morphologic and phenotypic analyses.

(37) Cell proliferation was estimated on days 7 and 10, before the occurrence of proplatelet extension. On day 7, there was a 6.7±1.6 fold and 4.2±1.2 fold expansion of the total nucleated cells (mean±SEM, n=8) in the absence and presence of SR1, respectively (FIG. 1B). From day 7 to day 10 the number of cells increased similarly, 2.3 and 2.5 times, in untreated and SR1-treated cultures, respectively. Therefore, SR1 did not promote cell proliferation under our culture conditions.

(38) We then evaluated the effect of SR1 on the maintenance of progenitors by following the evolution of CD34 expression. CD34 positivity was preserved in control and SR1-treated cells during the expansion step, with only a 16.8 and 8.3% decrease in positivity on day 7, respectively (FIG. 1C). On day 10, following passage in the presence of TPO alone, the proportion of CD34.sup.+ cells dropped to 40.7% in control cultures whereas 71.6% remained positive after SR1 treatment. Thus, SR1 maintained a more progenitor-like phenotype following transfer of the cells into media containing only added TPO.

(39) SR1 Increases the Production of Proplatelet-Bearing MKs and Platelet-Like Elements

(40) In control cultures, proplatelet extension was first observed on day 10 and culminated on day 14 when 11.5±4.5% of the MKs exhibited proplatelets (FIG. 2A). Remarkably, this proportion tripled after SR1 treatment (34.6±2.1%, mean±SEM, n=4) and this resulted in an increased production of platelet-like elements. Whereas under control conditions 7.92±3.25 platelet sized particles were counted per cell seeded on day 7 (FIG. 2B), the number of platelet particles increased about 3 fold after addition of SR1 (20.72±5.19). These results indicated that SR1 not only sustained progenitor potential but also greatly improved MK maturation. On the contrary, when SR1 was replaced by FICZ, a strong agonist of the AhR, there was a dramatic decrease in the proplatelet extension of MKs and the production of platelet-like elements (0.20±0.04 platelets/seeded cell). Such results strongly suggested that AhR blockade is at the origin of the increased platelet production in the presence of SR1. The antagonist activity of SR1 was confirmed on the inhibition of the expression of its downstream target CYP1B1 in a day 7 culture as measured by qPCR (579.8±40.8 vs 2.5±0.8 arbitrary units in control and SR1 treated cells, respectively; means±SEM, n=3).

(41) SR1 Promotes the Expansion of a CD34.sup.+CD41.sup.dim Population

(42) The above findings pointed to a dual effect of SR1 as it sustained CD34 expression and also improved MK maturation. Since CD41 is a specific marker of MKs, we evaluated its evolution in parallel with that of CD34. On day 7, a similar high proportion of CD34.sup.+ cells had acquired CD41 positivity with respectively 60 and 69% of the cells being CD34.sup.+CD41.sup.+ in control and SR1-treated cultures (FIG. 3A). Passage in the presence of TPO alone led to a significant loss of CD34 positivity in control cultures, with only 32% of the cells being CD34.sup.+CD41.sup.+ on day 10. In contrast, a high proportion (55%) remained double positive for CD34 and CD41 in SR1-treated cultures. Remarkably, a large fraction of these cells (37% of the total cells vs 17% in controls) exhibited a CD41.sup.dim phenotype (region R2) (FIG. 3B). The CD41.sup.dim population (R.sub.2) comprised cells of decreased size as compared to those with a higher level of CD41 (R.sub.1), as evidenced by their FSC properties (FIG. 3C), indicating a lower degree of MK differentiation. This was confirmed by the ploidy analysis since CD34.sup.+CD41.sup.dim cells were mostly 2n−4n (FIG. 4).

(43) CD34.sup.+CD41.sup.dim Cells have a High Capacity to Produce Proplatelets and Platelet-Like Particles

(44) Addition of SR1 in the two-step culture protocol resulted in an increased production of proplatelet-bearing MKs and platelet-like elements (FIG. 2A-B). We therefore investigated whether this was related to the expansion and particular properties of the CD34.sup.+CD41.sup.dim population. CD34.sup.+CD41.sup.dim cells from a day 10 culture with SR1 were sorted by flow cytometry and cultured for 7 days in a TPO-containing medium supplemented or not with SR1 (FIG. 5A). An unprecedented high proportion of MKs reached the proplatelet stage (91.0±2.4%) when CD34.sup.+CD41.sup.dim cells were grown in the presence of SR1 (FIG. 4B). Much lower frequencies were observed when these same cells were cultured in the absence of SR1 (10.0±6.6%) (FIG. 5B). The increased proplatelet yield led to a 6.8 fold enhanced production of platelet-like elements in CD34.sup.+CD41.sup.dim cells cultured with SR1 as compared to without SR1 (52.06±8.79 vs 7.68±0.81 platelets/seeded cell, respectively) (FIG. 5C). These results indicated that the CD34.sup.+CD41.sup.dim population expanded in the presence of SR1 has a strong potential to produce proplatelet-bearing MKs which are prone to release platelets.

(45) Co-Culture with MSCs Also Promotes the Emergence of a CD34.sup.+CD41.sup.dim Population

(46) Bone marrow-derived stromal cells can maintain hematopoietic stemness, secrete cytokines and favor MK maturation (Pallotta I et al., PloSone. 2009; 4:e8359; Cheng L et al., Journal of cellular physiology. 2000; 184:58-69) and could provide a favorable milieu for the emergence of an MK precursor. CD34.sup.+ cells were cultured in a two-step protocol on preformed monolayers of human mesenchymal stromal cells (hMSCs) isolated from human bone marrow (FIG. 6A). Co-culture with hMSCs did not significantly modify cell proliferation (FIG. 6B) but resulted in increased production of proplatelet-bearing MKs (data not shown) and platelet-like particles on day 14 (7.9±4.5 vs 18.2±4.9 platelets/cell seeded on day 7) (FIG. 6C).

(47) We investigated whether the effects of MSCs might be mediated by a pathway downstream of the AhR. Addition of the AhR agonist FICZ (Boitano A E et al., Science. 2010; 329:1345-1348) reduced the proportion of CD34.sup.+CD41.sup.dim cells (data not shown) and prevented the increase in platelet production (FIG. 7A). In addition, co-culture of CD34 cells with MSCs resulted in a profound decrease (>90%) in CYP1B1 transcripts, reproducing the effect of SR1 (FIG. 5E), an effect that was reversed by the addition of FICZ (FIG. 7B). These results indicated that MSCs similarly to SR1 promote MK maturation and platelet production by acting on the AhR.

(48) This response resembled that obtained with SR1, which prompted us to determine the CD34/CD41 phenotype of the cells. A population with the CD34.sup.+CD41.sup.dim profile was clearly apparent by day 10 of co-culture (FIG. 8A) and represented 30.37±1.98% of the total cells, as compared to 18.95±1.75% in control cultures without MSCs (FIG. 8B). This population was of low ploidy (FIG. 8C).

(49) We then sought to determine whether i) MSC- and SR1-derived CD34.sup.+CD41.sup.dim cells had the same potential to produce mature MKs and ii) co-culture with MSCs or in the presence of SR1 similarly favored this maturation. CD34.sup.+ cells were cultured with SR1 or on MSCs and the corresponding CD34.sup.+CD41.sup.dim cells were sorted on day 10 (FIG. 9). These cells were then subcultured for 7 days with TPO, with TPO and SR1 or with TPO and MSCs. The results showed that MSC-derived CD34.sup.+CD41.sup.dim cells exhibited an increased capacity to produce proplatelet-bearing MKs when cultured in the presence of SR1 (FIG. 9A, left panel), but with lower efficiency than SR1-derived cells (FIG. 9B, left panel) (49.5±10.5% vs 91.0±2.4%, respectively, n=4). In addition, co-culture with MSCs enhanced the MK maturation of both MSC-derived (FIG. 9A) and SR1-derived (FIG. 9B) CD34.sup.+CD41.sup.dim cells (53.3±10.7% vs 67.5±12.6%, respectively) compared to culture with TPO alone (%). Similar profiles were observed for the capacity to liberate platelet-like particles (FIGS. 9A-B, right panels). Thus, co-culture with MSCs phenocopied the responses obtained by adding SR1 to cell cultures.

(50) Altogether, it is therein reported the identification and enrichment of a discrete population of adult hematopoietic progenitors primed for MK differentiation which can efficiently mature to proplatelet-bearing MKs. This population, identified by means of its CD34.sup.+CD41.sup.dim signature, was amplified when adult CD34.sup.+ cells were cultured in the presence of SR1, an antagonist of the AhR, or an MSC monolayer. Culture with SR1 or MSCs, in addition to promoting the appearance of this MK progenitor, greatly improved the yield of proplatelet-producing MKs and the release of platelet-like elements.

(51) Several features of the CD34.sup.+CD41.sup.dim population identified here in the human system, such as the small size and low ploidy of the cells and their high capacity to mature into pure MKs able to efficiently extend proplatelets, appear to correspond to the definition of a platelet-biased progenitor. Its distinctive phenotype combines a CD34.sup.+ progenitor signature with intermediate or dim expression of the CD41 megakaryocytic marker. CD41 positive cells have been described among human CD34.sup.+ cells isolated directly from bone marrow or after culture under MK promoting conditions (Debili N. et al., Blood. 1992; 80:3022-3035; Dercksen M W et al., Blood. 1995; 86:3771-3782). However, these populations did not fully recapitulate the CD34.sup.+CD41.sup.dim phenotype since they appeared to express higher levels of CD41 and were highly polyploid and unable to proliferate (Dercksen M W et al., Blood. 1995; 86:3771-3782). CD34.sup.+CD41.sup.+ cells have also been observed after co-culture of bone marrow-derived CD34.sup.+ cells on hMSCs without TPO, but no evidence was provided for a distinct CD41.sup.dim subpopulation (Cheng L. et al., Journal of cellular physiology. 2000; 184:58-69). Cells with a CD34.sup.+CD41.sup.low phenotype representing a very minor population were recently reported in cultures derived from peripheral blood but were not characterized further. The CD34.sup.+CD41.sup.dim population was similarly of low frequency in our standard cultures (FIG. 3A) and only became apparent upon addition of SR1 or co-culture with MSCs. A CD31.sup.+CD34.sup.+CD41.sup.+ megakaryoblastic population resembling the cells described here was recently observed in reprogrammed iPS cells cultured in a three-step serum-free system. This population appeared to express low levels of CD41 and was negative for CD42.

EXAMPLE 2

(52) Materials and Methods

(53) Peripheral blood CD34.sup.+ cells were isolated as described above in the section “Isolation of CD34.sup.+ cells” and cultured in the presence of SR1 (1 μM) as described above in the section “MK differentiation in culture” of example 1.

(54) The cells recovered on day 10 were incubated with a mixture of Alexa-488-conjugated anti-CD41 (ALMA.17), phycoerythrin (PE)-Cy7-conjugated anti-CD34 monoclonal antibodies and phycoerythrin (PE)-CD9 (mAbs; BD Biosciences) for 30 minutes at 4° C. They were then incubated for 2 minutes in phosphate-buffered saline containing 7-aminoactinomycin D (2.5 μg/mL) to select viable cells.

(55) The cells were first subdivided into CD34.sup.+CD9.sup.− progenitors. Cell sorting using CD9.sup.− excludes CD41.sub.high cells because only CD9+ cells are CD4.sub.high as shown in FIG. 11. The population of CD34.sup.+CD9.sup.− progenitor cells (which thus does not comprise CD41.sup.high cells) was then fractionated into MK progenitors according to the FSC/CD41.sup.+ expression. The only CD41.sup.+ present in the cell population are CD41.sup.dim therefore allowing to gate on the population of interest of CD34.sup.+CD9.sup.−CD41.sup.dim cells. Megakaryocytic precursors were then sorted at 500 cells/s using a fluorescence-activated cell sorter (FACS) Aria II flow cytometer (Becton Dickinson, Mountain View, Calif.). The sorted CD34.sup.+CD9.sup.−CD41.sup.dim cells were then counted and seeded at 4×10.sup.4/mL in 48-well plates in StemSpan medium containing TPO, with or without SR1, for 7 days.

(56) Results

(57) The population previously described as being CD34.sup.+CD41.sup.dim can be also characterized as CD34.sup.+CD9.sup.−CD41.sup.dim. In particular, CD34.sup.+CD9.sup.−CD41.sup.dim represents a subpopulation of CD34.sup.+CD41.sup.dim, wherein the population CD34.sup.+CD9.sup.−CD41.sup.dim represents 60% of the total population of CD34.sup.+CD41.sup.dim cells. The differentiation potential of CD34.sup.+CD41.sup.dim cells compared to CD34.sup.+CD9.sup.−CD41.sup.dim cells was functionally examined. CD34.sup.+CD9.sup.−CD41.sup.dim cells have a platelet release that is increased by 1.8 fold compared to CD34.sup.+CD41.sup.dim cells.