Human Fetal Liver: An In Vitro Model of Erythropoiesis

We previously described the large-scale production of RBCs from hematopoietic stem cells (HSCs) of diverse sources. Our present efforts are focused to produce RBCs thanks to an unlimited source of stem cells. Human embryonic stem (ES) cells or induced pluripotent stem cell (iPS) are the natural candidates. Even if the proof of RBCs production from these sources has been done, their amplification ability is to date not sufficient for a transfusion application. In this work, our protocol of RBC production was applied to HSC isolated from fetal liver (FL) as an intermediate source between embryonic and adult stem cells. We studied the erythroid potential of FL-derived CD34+ cells. In this in vitro model, maturation that is enucleation reaches a lower level compared to adult sources as observed for embryonic or iP, but, interestingly, they (i) displayed a dramatic in vitro expansion (100-fold more when compared to CB CD34+) and (ii) 100% cloning efficiency in hematopoietic progenitor assays after 3 days of erythroid induction, as compared to 10–15% cloning efficiency for adult CD34+ cells. This work supports the idea that FL remains a model of study and is not a candidate for ex vivo RBCS production for blood transfusion as a direct source of stem cells but could be helpful to understand and enhance proliferation abilities for primitive cells such as ES cells or iPS

Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene-Driven Myeloid Leukemia.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631

Prise en charge biologique des leucémies aiguës myéloïdes (dix ans d'expérience à l'Hôpital Trousseau)

Les leucémies aiguës myéloïdes (LAM) représentent 15 à 20% des leucémies aiguës de l'enfant. Le diagnostic initial de la maladie repose sur des données biologiques précises regroupant la morphologie cellulaire, la classification FAB (French-American-British), l'immunophénotypage, la cytogénétique (classification MRC : Medical Research Council) et plus récemment la biologie moléculaire. La prise en charge des patients atteints de LAM s'est considérablement améliorée. Alors que la mortalité était très élevée dans les années 60, la survie globale est estimée actuellement à 60%.L'objectif principal de ce travail est d'analyser tous les patients traités pour une LAM de novo à l'hôpital Trousseau entre les années 1993 et 2003.Cette étude rétrospective monocentrique regroupe des données cliniques et biologiques au diagnostic de la maladie. Nous avons recherché dans notre cohorte les facteurs pronostiques tels que l'âge, l'hyperleucocytose, la présentation morphologique et les remaniements chromosomiques. Nous avons regardé l'impact de ces différents facteurs sur le devenir des patients en terme de survie globale, de rechute et de survie sans évènement. Nous avons également voulu comparer notre population aux études récentes de la littérature. Nous avons pu confirmer dans notre série l'inrérêt des classifications FAB et MRC avec un pronostic très péjoratif des LAM7-FAB et du groupe cytogénétique MRC3. Nous proposons ainsi 3 groupes pronostiques très différents : (1) groupe de bon pronostic : les leucémies avec les anomalies cytogénétiques suivantes t (8 ; 21), t (15 ; 17), inv (16) correspondant au groupe MRC1, (2) groupe de mauvais pronostic : les leucémies avec anomalies cytogénétiques impliquant les chromosomes 5 et 7, les caryotypes complexes et les leucémies mégacaryoblastiques, et (3) groupe de pronostic intermédiaire : toutes les leucémies n'appartenant à aucun de deus groupes précédents.PARIS7-Xavier Bichat (751182101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Germline RUNX1 variants in paediatric patients in a French specialised centre

Abstract Familial platelet disorder with associated myeloid malignancy (FPD‐MM; OMIM 601399) is related to germline RUNX1 mutation. The pathogenicity of RUNX1 variants was initially linked to FPD‐MM phenotype, but the discovery of new variants through the expansion of genetic explorations in leukaemia is questioning this assertion. In this study, we add 10 families with germline RUNX1 variant explored at Armand Trousseau Hospital for leukaemia diagnosis or thrombocytopenia, to the 259 described so far. Detailed description of their personal and family history of haematological pathologies allows identifying three phenotypes related to germline RUNX1 variants: thrombocytopenia and/or malignant haematological disease with family history of haematological diseases, thrombocytopenia with no family history of haematological diseases and acute lymphoblastic leukaemia (ALL) with no family history of haematological diseases. In the latter phenotype, ALL characteristics involving RUNX1 suggest the implication of germline variants in the onset of the malignancy

Molecular signature of erythroblast enucleation in human embryonic stem cells

International audienceWhile enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs

A cytometric study of the red blood cells in gaucher disease reveals their abnormal shape that may be involved in increased erythrophagocytosis

International audienceGaucher disease is a sphingolipidosis caused by a deficiency of the enzyme glucocerebrosidase. Macrophages transform into pathogenic Gaucher cells following the phagocytosis of red blood cells (RBCs) and subsequent accumulation of glucosylceramide. Enhanced erythrophagocytosis is one feature of the disease indicating abnormal macrophage-RBC interactions. We hypothesized that the erythrophagocytosis observed in Gaucher disease may be at least partly due to abnormalities in the RBCs themselves. Methods: To investigate this hypothesis, we used flow cytometry FSC/SSC to study RBCs sampled from seven patients with Gaucher disease in terms of their shape and the expression of markers of senescence and phagocytosis. Cells from two of the seven patients were evaluated before and 9 months after the start of enzyme-replacement therapy. Results: Untreated patients were found to have abnormal flow-cytometry profiles suggesting an alteration of Gaucher RBC morphology. Scanning electron microscopy confirmed this finding by revealing many abnormally shaped RBCs. Whereas there was no evidence of desialylation of membrane glycoconjugates or phosphatidylserine exposure, RBC viability (calcein-AM test) and CD47 expression were reduced. These anomalies found in RBCs sampled from two patients before treatment, were no longer present after a 9 month-long enzyme-replacement therapy. Conclusions: We report on previously overlooked alterations of Gaucher RBCs that may facilitate erythrophagocytosis in untreated patients. Their potential role in the anemia, the excess of aggregation and rheological anomalies associated with Gaucher disease must now be addressed. RBC anomalies may take part in the abnormal crosstalk between RBCs and macrophages leading to the accumulation of Gaucher cells. (C) 2010 International Clinical Cytometry Societ