Search

The most frequent oncogenic activation events characterized in childhood T acute lymphoblastic leukemia (T-ALL) result in the transcriptional activation of genes coding for transcription factors. The main genes are TAL1/SCL, a member of the basic region helix-loop-helix gene family, and HOX11L2, a member of the homeobox-containing protein family. To gain insight into the pathogenesis of this type of hematologic malignancy, we analyzed 28 T-ALL samples. SIL-TAL1/SCL fusion was detected in 6 patients; expression of HOX11L2 was observed in 6 patients and of HOX11 in 3 patients. With one exception, these activations did not occur simultaneously in the same patients, and they allowed the subclassification of 50% of the patients.SIL-TAL1 fusion was detected in association withHOX11 expression in one patient and with a t(8;14) (q24;q11) in another. High expression of LYL1,LMO2, or TAL1 was observed mainly in samples negative for HOX11L2 expression. HOX11L1 andHOX11 expression were observed in one instance each, in the absence of detectable chromosomal abnormality of their respective loci, on chromosomes 2 and 10, respectively. HOX11L2 expression was associated with a chromosome 5q abnormality, the location of theHOX11L2 locus in each case tested. Finally, our data show that HOX11L2 expression was a suitable marker for minimal residual disease follow-up and was significantly associated with relapse (P = .02).

Background: The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients. Methods: Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH. Results: In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients. To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions. In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events. Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS). We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036). In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p Conclusion Our study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. Disclosures No relevant conflicts of interest to declare.

This study characterizes a new congenital thrombocytopenia with mild hemorrhagic tendency occurring in a woman and her child with the following features. We found a deletion of the distal part of one chromosome 11 [del(11)q23.3-->qter] that was detected by cytogenetic analysis and confirmed by chromosome painting in the two patients and also an increased number of bone marrow megakaryocytes (MKs), including numerous micromegakaryocytes (mMKs) associated with a normal platelet life span. A normal number of MK colonies in culture was observed with one third of them containing a few large MKs; however, these were always associated with mMKs identified by immunologic staining. A massive cell lysis was observed at the end of the maturation. Fifteen percent of the platelets in the peripheral blood showed giant alpha-granules resulting from the fusion of alpha-granules. These giant granules, which appeared in red on giemsa stain, had a mean diameter of 1.5 microns and showed all markers (detected at electron microscopy by immunogold method) of matrix and alpha-granule membrane, ie, von Willebrand factor, fibrinogen, CD41, CD62P (P-selectin); however, they differed from lysosomes because acid phosphatases were not present. These giant alpha-granules were unable to release their contents after stimulation by thrombin, in contrast to platelets with normal morphology. Abnormalities in bone marrow MK maturation that were detected at the electron microscopic level and that led to lysis of numerous MKs were responsible for thrombocytopenia and were similar in both patients. MK abnormalities are probably the consequence of the chromosome aberration. ETS 1 and FLI, two proto-oncogenes that appear to be essential with GATA1 for the normal expression of MK-specific genes, map to 11q23-q24 and are, thus, deleted in this thrombocytopenia. In conclusion, the association of all these abnormalities constitutes a new familial platelet disorder and may present a valuable model for exploring the role of some genes involved in the regulation of thrombopoiesis.

*the first two authors are co-first authors Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process. Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern. To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins. Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Introduction Acute myeloid leukemia (AML) is an aggressive malignancy caused by the accumulation of multiple oncogenetic mutations occurring in a single lineage of hematopoietic progenitors. AML is rare in children and the mutations found are partially different from those in adults, and for some with a lower frequency. Thus, clonal evolution leading to pediatric AML may be specific, and has not been described yet. Methods To define clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trio of specimens (diagnosis, germline and relapse) in a 9-years old girl presenting AML FAB M5a with t(9;11)(p22;q23) MLL-AF9 and trisomy 8. At diagnosis, we focused on 3 non-silent somatic mutations candidate for leukemogenesis process, confirmed by Sanger method: EED (R355*), GSDMC (R40*) and ELK1 (3’ UTR). In the same time, we performed cell cultures from bone marrow mononucleated cells at diagnosis. CD34 and CD38 cells were cultured either in liquid long term culture medium (LTC IC) or methylcellulose medium. Results: A total of 512 colonies were collecte. Our 3 interest mutations and trisomy 8 were tracked by allele-specific PCR, and MLL rearrangement detected by FISH, individually in 267 from the 512 colonies. Exploitable results were found in 164 colonies. Through these results in the different cell populations, we were able to establish the clonal architecture at diagnosis. MLL-AF9 fusion and EED mutation were found together as the first concomitant occurring events in the leukemic clone. Then genotyping of the colonies demonstrated that ELK1 mutation, GSDMC mutation, and trisomy 8 were successively acquired. Additional later mutations such as ASXL1 (frameshift), PTPN11 (E76K), EMP2 (3’UTR) and DGCR14 (P314S) were detected in the relapse sample. Discussion The 3 mutations studied in the colonies may impact the progression of the leukemic clone by dysregulating several cellular pathways and networks. First, EED is an essential non-catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates gene silencing through catalysis of histone H3K27 methylation. PRC2 is known to be enhanced in solid neoplasms such as prostate cancer. On the contrary, in myeloid malignancies and myelodysplasic syndromes, it has been recently demonstrated that mutations involving PRC2 subunits (EED, SUZ12 and EZH1/2) were hypomorphic. These loss-of-functions mutations were responsible for chromatin relaxation and induced transcription of genes promoting self-renewal such as HOXA9. Nevertheless, recent sh-RNA studies in a murine model of MLL-AF9 leukemia demonstrated that residual PRC2 enzymatic activity after EED mutation is needed to unable leukemia growth. These data are coherent with our finding that EED mutation is an early event in leukemogenesis, in cooperation with MLL-AF9 rearrangement. Secondly, ELK1 is targeted by RAS-MAPK pathway, thus its mutation can confer an increased proliferation potential when acquired by the leukemic clone, after its maturation has been blocked and its self-renewal increased through previous MLL rearrangement and EED mutation. Finally, GSDMC may be implicated in monocyte count regulation, and mutated in other neoplasms such as melanoma. As a consequence, it is likely that its mutation occurs lately in the evolution of the monoblastic leukemic clone of our patient. The latest event in the clonal evolution in our patient at diagnosis is the acquisition of trisomy 8. Conclusion This study highlights the clonal evolution in one pediatric AML, and paves the way for further studies to better understand clonal evolution in children. Elucidating, the succession and the cooperation between driver and secondary mutations, is important for both understanding leukemogenesis and developing innovative therapeutic agents targeting founding anomalies in the leukemic clone at its most precocious stage. Moreover, discovering clonal architecture also unable to find new minimal residual disease markers to assess the therapeutic response and risk stratification. Disclosures No relevant conflicts of interest to declare.

Abstract SCI-39 The generation of red blood cells (RBCs) in vitro using biotechnologies could represent an interesting alternative to classical transfusion products, in that it would combine adequate supplies with the specific production of blood products of a particular phenotype and the reduction of infection risks. This presentation will review how it is now possible to obtain in vitro complete maturation of the erythroid line to the stage of enucleation, starting from hematopoietic stem cells (HSCs) from peripheral blood, bone marrow or umbilical cord blood, or embryonic stem cells or adult pluripotent stem cells (induced pluripotent stem cells, iPSCs). This presentation will discuss how the functionality of cultured human RBCs (cRBCs) is settled in terms of deformability, hemoglobin maturation, oxygen carrying capacity, enzyme content, and terminal maturation from the reticulocyte stage to mature RBC after infusion into the NOD/SCID mouse model. The clinical feasibility of this concept has recently been demonstrated by reporting that cRBCs generated in vitro from peripheral HSCs under GMP conditions encounter in vivo the conditions required for their maturation and that they persist in the circulation for several weeks in humans. These data have established the proof of principle for transfusion of in vitro-generated RBCs and the pathway toward new developments in transfusion medicine. The most proliferative source of stem cells for generating cRBCs is cord blood, but it is limited in terms of HSCs and is dependent on donations. Pluripotent stem cell technology represents a potentially unlimited source of RBCs and opens the door to the development of a new generation of allogeneic transfusion products. Because iPSCs can be selected for a phenotype of interest, they are obviously the best candidate for organizing complementary sources of RBCs for transfusion. It is established that only three human iPSC clones would have been sufficient to match more than 99 percent of the patients in need of RBC transfusions. As a whole, a very limited number of RBC clones would provide for the needs of most alloimmunized patients and those with a rare blood group. Generating cRBCs from iPSCs has been done but needs to be optimized to lead to a clinical application in blood transfusion. Several crucial points remain to be resolved, notably, the choice of the initial cell type, the method of reprogramming (i.e., to ensure the safety of the iPSCs and to ensure their clinical grade), the optimization of the erythrocyte differentiation, and the definition of GMP conditions for industrial production. Assuming that in vitro large-scale cultured RBC production efficiently operates in the near future, this presentation will highlight the potential applications for alloimmunized patients and those with a rare blood group. Disclosures: No relevant conflicts of interest to declare.

A total of 125 acute leukemia adult patients were autografted with bone marrow (BM) purged by mafosfamide (ASTA Z) during the period of January 1983 to January 1993. The median follow-up period was 64 months (range, 3 to 126). There were 84 acute myeloblastic leukemias (AMLs) and 41 acute lymphoblastic leukemias (ALLs). At time of autologous BM transplantation (ABMT); 64 AMLs were in first complete remission (CR1), and 20 were in second CR (CR2); 35 ALL were in CR1, and 6 were in CR2. The median age of the patients was 33 years (range, 16 to 55). The median interval between achieving CR and autografting was 5 months (range, 1.3 to 23). The pretransplant regimen consisted of cyclophosphamide (120 mg/kg) and total body irradiation. All patients were grafted with autologous BM treated in vitro with mafosfamide used at levels individually adjusted in 95 patients and at a standard dose in 30 patients. The initial richness in granulomacrophagic progenitors (CFU-GM) of the harvested BMs was 5.16 x 10(4) CFU-GM/kg (range, 0.55 to 33). After mafosfamide purging, the residual CFU-GM number was 0.021 x 10(4)/kg (range, 0 to 1.78). The probability of successful engraftment was significantly higher and the time to engraftment was significantly shorter in ALL. Of 33 patients grafted with BM containing no residual CFU-GM, those with AML (n = 22) had platelet recoveries that were significantly longer than those for AML patients receiving BM with residual CFU-GM. At 8 years, patients autografted in CR1 for AML and ALL had a leukemia-free survival (LFS) of 58% and 56%, respectively, with a relapse incidence (RI) of 25% and 37%, respectively. Patients autografted in CR2 for AML had an LFS of 34% and an RI of 48% at 5 years. The incidence of late relapses was significantly higher in ALLs. By multivariate analysis, four factors were found to influence favorably engraftment in addition to a diagnosis of ALL, a younger age, ABMT performed in CR1, the adjusted dose technique of purging, and a shorter interval from CR to ABMT. Two factors were correlated with a better outcome. (1) The LFS was significantly higher and the transplant-related mortality significantly lower in patients who received richer BM. (2) The RI was significantly lower in patients autografted within 150 days from CR. Our results reinforce the view that ABMT is one approach to improve the outcome of adult patients with acute leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Disseminated nasal-type natural killer (NK) cell lymphoma is an aggressive disease with very poor prognosis. The usual chemoresistance of this disease led us to explore the possible role of the transcription factor STAT3 in oncogenesis. For this, we established and characterized a continuous interleukin (IL)2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK cell lymphoma. MEC04 cells phenotype was CD3−, CD7+, TCR−, CD16−, CD56+bright, p58−, p70−, NKP−, and NKG2A+, they harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-γ, IL-10 and vascular-endothelium growth factor in vitro, suggesting that malignant cells arise from the CD56+bright regulatory NK cell population. Injection of MEC04 cells to NOD/SCID mice led to a fatal multiorgan infiltration by malignant cells. We show by immunohistochemistry that STAT3 is phosphorylated in Y705 dimerization residue in MEC04 and YT cell lines, and on biopsies from 6/6 patients with nasal-type NK cell lymphoma, suggesting that STAT3 may be oncogenic in this disease. By contrast, Y705 residue was not phosphorylated on 2 patients with hepatosplenic γδT-cell lymphoma. By confocal microscopy, we showed that STAT3 was located in nucleus in MEC04 cells. Y705 STAT3 phosphorylation involved JAK2, since exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation, and correlated in a dose-dependent growth inhibition at 24 hours and 48 hours. By using transducible TAT-STAT3b or TAT-STAT3Y705F recombinant proteins (a dominant-negative form of STAT3 [STAT3b isoform], and a STAT3 protein mutated on Y705 residue which prevents STAT3 dimerization [STAT3Y705F]), we were able to demonstrate that inhibition of STAT3 activation increased mortality and decreased proliferation by more than 50 p. cent at 24 hours and 48 hours, which correlated with decreased expression of 2 STAT3 target genes (Bcl-XL and c-Myc). These results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK cell lymphomas, and may thus represent a promising therapeutical target.

INTRODUCTION. Since the discovery of the cryptic t(12; 21) translocation, many secondary genetic abnormalities have been described in association with TEL-AML1 fusion gene. Extensive studies of karyotypes in a few series of TEL-AML1 positive leukaemia revealed a heterogeneous pattern of chromosomal abnormalities. Numerical and structural abnormalities are often present together. The modal chromosome number does not exceed 49 so that high hyperdiploidy and TEL-AML1 fusion are so far considered mutually exclusives. Here we reported that such association is found in a small group of patients and that relapse may still occur in those patients despite the good prognostic impact commonly attributed to each lesion. PATIENTS and METHODS. Between April 1994 and April 2006, 105 children were consecutively diagnosed with TEL-AML1 positive B-ALL. TEL-AML1 expression was detected by RT-PCR in Bone Marrow (BM) diagnostic samples and the t(12;21) explored by FISH with LSI TEL-AML1 dual-color probe (Vysis) in cases with low level of fusion gene expression or lacking molecular study. Conventional cytogenetics with G and R banding was performed on BM cells after overnight and 24 hours culture RESULTS and DISCUSSION. We detected numerical and/or structural abnormalities in 82/105 (78%) of the karyotypes. Aneuploidy alone was found in 4/105(3.8%): two cases had an extra chromosome 21, one an extra chromosome 10 and one lost 1 chromosome X. Structural abnormalities alone were present in 18/105 (17.1%) and up to 58 /105 (55.2%) presented both. The most frequent structural change was observed on chromosome 12p13 (50% of all structural abnormalities) whereas the most frequent chromosome gain was +21 (14%) and +10 (6.6%). Interestingly, in four cases (3.8%) we detected high hyperdiploidy with classical supplementary chromosomes; in two of these cases a partial trisomy of chromosome 1 was also present. Karyotypes are presented: Pt1: 52,XX,+10,+16,+18,+21,+21,+22 [22]/46, XX [4] Pt 2: 54,XY,+X,+4,+ 6?[del(6q)],+9,+14,+15,+18,+21,+21,-22 [17]/46,XY [3] Pt 3: 55,XX,+X,dup(1)(q21q31),+4,+6,+10,+14,+17,+18,+21,+21 [1]/56,idem+19 [4]/56,idem,+14 [5] / 46,XX [10] Pt 4: 7,XX,+der(X)(t(X;1)(q26;q12),+4,+5,+6,+8,+10,+14,+17,+18,+21,+21 [17]/46,XX [3] Pt1 present CNS relapse at 24 months follow up without involvement of BM which tested negative for TEL-AML1 expression. Patients 2,3,4 are in continuous complete remission at respectively 6, 6 and 4 years follow up. Since t(12;21) translocation and high hyperdiploidy seem to be primary events in leukaemia our observation raises the question if the two lesions coexist in a same cell or are independent events in different clones. In two cases (Pt 3,4), FISH analysis indicated the presence of TEL-AML1 gene fusion in 7% and in 5% of nuclei respectively. The distribution and the number of the signals on AML1 locus let think that t(12;21) occurred independently from hyperdiploidy. Microarrays studies have revealed distinct gene expression signatures associated to TEL-AML1 fusion and hyperdiploidy over 50 chromosomes, our observation points out the existence of cases which could be difficult to assign to one or the other of these classes.

The aim of our study was to determine whether ex vivo expansion of umbilical cord blood (UCB) progenitor cells induces changes in their capacity to generate immune cells. CD34+ CB cells were cultured for 14 days with SCF, FLT3-l, TPO and G-CSF, inducing a total cells, CD34+ and LTC-ICs increase of 1500, 120 and 8 fold respectively. Non expanded (d0) and 14-day expanded (d14) CD34+ cells were compared for their capacity to produce T lymphocytes (TLs) using the fetal thymus organ culture system and DCs generated from d0 and d14 CD34+ cells were compared for their differentiation, phenotype and function. Total percentages of CD4+, CD4+CD8− and CD4+CD8+ TLs obtained from d0 and d14 CD34+ cells were comparable. In both fractions, most of the CD4+ T cells co-expressed iCD3 but a lower proportion of d14 derived TLs expressed sCD3. However, there was no significant difference between d0 and d14 derived TLs in term of Vb chain representation, all TCR-Vb chains examined being represented in each case. These data indicate that d0 and d14 CD34+ cells have a similar capacity to generate TLs and that expansion does not induced any skewing of the TCR-Vb repertoire. D0 and d14 CD34+ cells were next cultured with SCF, FL, GM-CSF and TNF-a to compare their capacity to differentiate into DCs. Similar percentages of CD1a+ DCs expressing the same levels of HLA-DR and co stimulatory molecules were obtained. DCs derived from d14 CD34+ cells were less potent to stimulate allogeneic TLs, but the pattern of cytokines produced by stimulated TLs was similar and no shift towards a predominant Th1 or Th2 response was observed. Moreover, in spite of a quantitative increase (15 fold) related to the CD34 pool amplification, we observed a decreased capacity (13-fold) of d14 cells to generate DCs compared to d0 CD34+ cells. Overall, these results indicate that ex vivo expansion of CD34+ cells doesn’t induce any major modification in T Lymphopoiesis capacity while alters somehow the capacity of the graft to generate DCs. We discuss in the context of UCB transplantation, the putative interest of co-infusion of expanded and non expanded fractions in view of improving myelopoiesis in the graft without subverting the immune reconstitution.

We report a technological approach permitting, for the first time, the massive (up to 2x106-fold cell expansion) and selective (100%) ex vivo production of mature RBCs (cRBCs) starting from CD 34+ cells from peripheral blood (PB), bone marrow (BM) or cord blood (CB) into mature red cells in three steps: firstly, cell proliferation and erythroid differentiation were induced in serum free media supplemented with SCF, IL-3 and Epo for 8 days. Secondly, cells were co-cultured with additional Epo alone on either the murine MS-5 stromal cell line or human mesenchymal cells for 3 days. In the third step, all exogenous factors were withdrawn and cells were incubated on a simple stroma for 4 to 10 days. These cultured erythroid cells (reticulocytes and mature RBCs) displayed characteristics identical to those of native cells, in terms of MCV, MCH, MCHC, enzyme content (G6PD and PK) and deformability. The nature of the Hb produced depended on both the origin of the CD34+ cells and the culture conditions. cRBCs derived from PB or adult BM contained adult Hb (95±1%) whereas cRBCs derived from CB contained essentially HbF (64±13%). As for native RBCs, Hb was able to fix and release oxygen. CFSE-labelled-reticulocytes ex vivo generated from leukapheresis were injected into NOD-SCID mice. The transfused reticulocytes were found in the circulation to the same extent as native RBCs and fully matured into RBCs. This methodology is applicable for fundamental analysis of the mechanisms of terminal erythropoiesis and hemoglobin synthesis. Moreover, large scale cRBCs production could be possible with such a protocol. It can therefore be extrapolated to a wide range of clinical applications in the field of gene therapy, infectious diseases and particularly transfusion medicine with a pointed interest for the generation of a cell population homogeneous in age, thus achieving the new concept of cultured RBCs transfusion.