PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells.

RNA-binding proteins (RBPs) have emerged as important regulators of invertebrate adult stem cells, but their activities remain poorly appreciated in mammals. Using a short hairpin RNA strategy, we demonstrate here that the 2 mammalian RBPs, PUMILIO (PUM)1 and PUM2, members of the PUF family of posttranscriptional regulators, are essential for hematopoietic stem/progenitor cell (HSPC) proliferation and survival in vitro and in vivo upon reconstitution assays. Moreover, we found that PUM1/2 sustain myeloid leukemic cell growth. Through a proteomic approach, we identified the FOXP1 transcription factor as a new target of PUM1/2. Contrary to its canonical repressive activity, PUM1/2 rather promote FOXP1 expression by a direct binding to 2 canonical PUM responsive elements present in the FOXP1-3' untranslated region (UTR). Expression of FOXP1 strongly correlates with PUM1 and PUM2 levels in primary HSPCs and myeloid leukemia cells. We demonstrate that FOXP1 by itself supports HSPC and leukemic cell growth, thus mimicking PUM activities. Mechanistically, FOXP1 represses the expression of the p21-CIP1 and p27-KIP1 cell cycle inhibitors. Enforced FOXP1 expression reverses shPUM antiproliferative and proapoptotic activities. Altogether, our results reveal a novel regulatory pathway, underscoring a previously unknown and interconnected key role of PUM1/2 and FOXP1 in regulating normal HSPC and leukemic cell growth.

Neuraminidase enhances in vitro expansion of human erythroid progenitors.

In spite of recent key improvements, in vitro mass production of erythrocytes from human stem cells is still limited by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, such progenitors are as scarce in the bone marrow as in peripheral blood. We used a two-step culture model of human cord blood-derived erythroid progenitors in the presence or absence of high-purity neuraminidase, in a serum-free, defined culture medium. Granulocytic and megakaryocytic progenitor cell expansions were also studied. We show that significant enhancement of erythroid cell generation is obtained when CD34(+) human hematopoietic progenitors are cultured in the presence of neuraminidase. Interestingly, in so doing, expanded red cell progenitors remained erythropoietin-dependent for further expansion and survival, and cells thus generated displayed a normal phenotype. Moreover, the activity of neuraminidase on these cells can be reversed by simple cell washing. Finally, growth of cells of the other myeloid lineages (granulocytes and megakaryocytes) is either decreased or unchanged in the presence of neuraminidase. This specific feature of neuraminidase, that of stimulation of human red cell progenitor proliferation, provides a safe technique for producing greater numbers of in vitro-generated red blood cells for both basic research and transfusion use.

HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4.

BACKGROUND: Expansion of hematopoietic stem cells represents an important objective for improving cell and gene therapy protocols. Retroviral transduction of the HoxB4 homeogene in mouse and human hematopoietic stem cells and hematopoietic progenitors is known to promote the cells' expansion. A safer approach consists in transferring homeobox proteins into hematopoietic stem cells taking advantage of the natural ability of homeoproteins to cross cell membranes. Thus, HOXB4 protein transfer is operative for expanding human hematopoietic cells, but such expansion needs to be improved. DESIGN AND METHODS: To that aim, we evaluated the effects of HOXC4, a protein encoded by a HOXB4 paralog gene, by co-culturing HOXC4-producing stromal cells with human CD34(+) hematopoietic cells. Numbers of progenitors and stem cells were assessed by in vitro cloning assays and injection into immuno-deficient mice, respectively. We also looked for activation or inhibition of target downstream gene expression. RESULTS: We show that the HOXC4 homeoprotein expands human hematopoietic immature cells by 3 to 6 times ex vivo and significantly improves the level of in vivo engraftment. Comparative transcriptome analysis of CD34(+) cells subjected or not to HOXB4 or HOXC4 demonstrated that both homeoproteins regulate the same set of genes, some of which encode key hematopoietic factors and signaling molecules. Certain molecules identified herein are factors reported to be involved in stem cell fate or expansion in other models, such as MEF2C, EZH2, DBF4, DHX9, YPEL5 and Pumilio. CONCLUSIONS: The present study may help to identify new HOX downstream key factors potentially involved in hematopoietic stem cell expansion or in leukemogenesis.

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells.

BACKGROUND: Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important. DESIGN AND METHODS: Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation. RESULTS: Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice. CONCLUSIONS: This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.

Concise review: Anemia caused by viruses.

Most of the viruses known to be associated with anemia in human tend to persistently infect their host and are noncytopathic or poorly cytopathic for blood cell progenitors. Infections with Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, human herpes virus 6 (HHV-6), B19 parvovirus, human immunodeficiency virus, hepatitis A and C viruses and the putative viral agent associated with non-A-G post-hepatitis aplastic anemia have been reported in association with anemia. Nevertheless, a direct cytotoxic effect on erythroid progenitors has been clearly demonstrated only for human parvovirus B19 and evocated for HHV-6. A major role for destructive immunity is strongly suspected in the pathogenesis of anemia associated with the other viral infections. Host genes play a role in the occurrence of virus-induced anemia in animal models, and there are some evidences that genetic background could also influence the occurrence of virus-associated anemia in human.

Efficient in vitro myogenic reprogramming of human primary mesenchymal stem cells and endothelial cells by Myf5.

BACKGROUND INFORMATION: The identification of a source of stem cells able to regenerate skeletal muscle was the goal of numerous studies with the aim to develop new therapeutic approaches for genetic muscle diseases or muscle injuries. A series of studies have demonstrated that stem cells derived from various tissues may have a role in the regeneration of damaged muscles, but this contribution is always very weak. Thus we established a project aiming to reprogramme non-muscle cells into the skeletal striated differentiation pathway. RESULTS: We transduced several human primary adult stem or progenitor cells using a recombinant lentivirus containing the coding sequence of the Myf5 gene considered as a master gene for the determination of skeletal striated muscle. These original results are the first demonstration of a myogenic conversion of human mesenchymal and endothelial cells by Myf5. CONCLUSIONS: The procedure described in the present paper could be used to develop new research protocols with the prospect of using these cells as therapeutic agents.

Leukemic stem cell persistence in chronic myeloid leukemia patients with sustained undetectable molecular residual disease.

Sustained undetectable molecular residual disease (UMRD) is obtained in a minority of patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors. It remains unclear whether these patients are definitively cured of their leukemia or whether leukemic stem cells (LSCs) persist in their BM. We have evaluated the presence of BCR-ABL-expressing marrow LSCs in 6 patients with chronic myeloid leukemia with sustained UMRD induced by IFN-α (n = 3), imatinib mesylate after IFN-α failure (n = 2), and dasatinib after imatinib intolerance (n = 1). Purified CD34(+) cells were used for clonogenic and long-term culture-initiating cell assays performed on classic or HOXB4-expressing MS-5 feeders. Using this strategy, we identified BCR-ABL-expressing LSCs in all patients. Interestingly, long-term culture-initiating cell assays with MS-5/HOXB4 stromal feeders increased detected numbers of LSCs in 3 patients. The relation between LSC persistency and a potential risk of disease relapse for patients with durable UMRD (on or off tyrosine kinase inhibitor therapy) warrants further investigation.

Exposure of human megakaryocytes to high shear rates accelerates platelet production.

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Chromatin modifications in hematopoietic multipotent and committed progenitors are independent of gene subnuclear positioning relative to repressive compartments.

To further clarify the contribution of nuclear architecture in the regulation of gene expression patterns during differentiation of human multipotent cells, we analyzed expression status, histone modifications, and subnuclear positioning relative to repressive compartments, of hematopoietic loci in multipotent and lineage-committed primary human hematopoietic progenitors. We report here that positioning of lineage-affiliated loci relative to pericentromeric heterochromatin compartments (PCH) is identical in multipotent cells from various origins and is unchanged between multipotent and lineage-committed hematopoietic progenitors. However, during differentiation of multipotent hematopoietic progenitors, changes in gene expression and histone modifications at these loci occur in committed progenitors, prior to changes in gene positioning relative to pericentromeric heterochromatin compartments, detected at later stages in precursor and mature cells. Therefore, during normal human hematopoietic differentiation, changes in gene subnuclear location relative to pericentromeric heterochromatin appear to be dictated by whether the gene will be permanently silenced or activated, rather than being predictive of commitment toward a given lineage.

A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

The HOXB4 homeoprotein differentially promotes ex vivo expansion of early human lymphoid progenitors.

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

PU.1/Spi-1 binds to the human TAL-1 silencer to mediate its activity.

The TAL-1/SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for primitive hematopoiesis and for adult erythroid and megakaryocytic development. Activated transcription of TAL-1 as a consequence of chromosomal rearrangements is associated with a high proportion of human T cell acute leukemias, showing that appropriate control of TAL-1 is crucial for the formation and subsequent fate of hematopoietic cells. Hence, the knowledge of the mechanisms, which govern the pattern of TAL-1 expression in hematopoiesis, is of great interest. We previously described a silencer in the 3'-untranslated region of human TAL-1, the activity of which is mediated through binding of a tissue-specific 40 kDa nuclear protein to a new DNA recognition motif, named tal-RE. Here, we show that tal-RE-binding activity, high in immature human hematopoietic progenitors is down regulated upon erythroid and megakaryocytic differentiation. This expression profile helped us to identify that PU.1/Spi-1 binds to the tal-RE sequences in vitro and occupies the TAL-1 silencer in vivo. By expressing a mutant protein containing only the ETS domain of PU.1 in human erythroleukemic HEL cells, we demonstrated that PU.1 mediates the transcriptional repression activity of the silencer. We found that ectopic PU.1 is not able to induce silencing activity in PU.1-negative Jurkat T cells, indicating that PU.1 activity, although necessary, is not sufficient to confer transcriptional repression activity to the TAL-1 silencer. Finally, we showed that the silencer is also active in TAL-1-negative myeloid HL60 cells that express PU.1 at high levels. In summary, our study shows that PU.1, in addition to its positive role in TAL-1 expression in early hematopoietic progenitors, may also act as a mediator of TAL-1 silencing in some hematopoietic lineages.

[Ex vivo expansion of human hematopoietic stem cells by passive transduction of the HOXB4 homeoprotein]. / Expansion ex vivo des cellules souches hématopoïétiques humaines par la transduction passive de l'homéoprotéine HOXB4.

Expansion of human hematopoietic stem cells (HSCs) is a challenge for cellular therapy. It currently relies on either the use of recombinant cytokines or transfer of transcription factor genes. Among these, the HOXB4 homeoprotein is of particular interest since it promotes the expansion of mouse HSCs without inducing leukemia. To prevent potential deleterious side effects associated with stable HOXB4 gene transfer into the cells, we took advantage of the ability of homeoproteins to passively pass through cell membranes. We have shown that, when co-cultured with stromal cells engineered to secrete HOXB4, human stem cells and immature progenitors clearly were expanded. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. The role that HOXB4 plays on stem cell expansion has also been tested on human lymphoid progenitors. We found that our model of protein transfer was also able to induce the expansion of the immature lympho-myeloid and pro-T/NK progenitors as well as of more mature NK progenitors. We then looked for synergistic activities between HOXB4 and other homeoproteins such as HOXC4. We found that HOXC4 was able to promote the expansion of human hematopoietic cells in vitro roughly as HOXB4 did and that the presence of both HOXB4 and HOXC4 molecules induced even higher expansion levels of these cells. Our method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

NACA is a positive regulator of human erythroid-cell differentiation.

We have previously identified the transcript encoding NACA (the alpha chain of the nascent-polypeptide-associated complex) as a cytokine-modulated specific transcript in the human TF-1 erythroleukemic cell line. This protein was already known to be a transcriptional co-activator that acts by potentiating AP-1 activity in osteoblasts, and is known to be involved in the targeting of nascent polypeptides. In this study, we investigate the role of NACA in human hematopoiesis. Protein distribution analyses indicate that NACA is expressed in undifferentiated TF-1 cells and in human-cord-blood-derived CD34(+) progenitor cells. Its expression is maintained during in vitro erythroid differentiation but, in marked contrast, its expression is suppressed during their megakaryocytic or granulocytic differentiation. Ectopic expression of NACA in CD34(+) cells under culture conditions that induce erythroid-lineage differentiation leads to a marked acceleration of erythroid-cell differentiation. Moreover, ectopic expression of NACA induces erythropoietin-independent differentiation of TF-1 cells, whereas downregulation of NACA by RNA interference abolishes the induction of hemoglobin production in these cells and diminishes glycophorin-A (GPA) expression by CD34(+) progenitors cultured under erythroid differentiation conditions. Altogether, these results characterize NACA as a new factor involved in the positive regulation of human erythroid-cell differentiation.

IFN regulatory factor-2 cooperates with STAT1 to regulate transporter associated with antigen processing-1 promoter activity.

Class I MHC complexes (MHC(I)) are essential in mediating immune response. The transport of antigenic peptides (TAP) to MHC(I) and the stable expression of MHC(I) on the cell surface require the presence of a dedicated TAP. In this study we report that IFN-gamma and thrombopoietin (TPO) strongly increase TAP1 protein expression in megakaryocytes, followed by an enhanced expression of MHC(I) on the cell surface. This expression parallels the enhanced TAP1 promoter activity and TAP1 mRNA expression, which are independent of protein synthesis. We also show that this cytokine-dependent expression of TAP1 transcripts depends on STAT1 and IFN regulatory factor-2 (IRF-2), but not on IRF-1, and provide evidence that IRF-2 constitutively binds to the TAP1 gene promoter and enhances TAP1 promoter activity. We show that IRF-2 forms a complex with STAT1 and the cytokine-responsive region of the TAP1 promoter in any TPO or IFN-gamma target cells tested. Interaction of IRF-2 and STAT1 on the promoter depends on the DNA-binding domain of IRF-2. Overall, our data indicate that TPO and IFN-gamma activate the expression of TAP1 via a new mechanism that involves functional cooperation between STAT1 and IRF-2 on the TAP1 promoter.

Hypoxia enhances human B19 erythrovirus gene expression in primary erythroid cells.

Human B19 erythrovirus replicates in erythroid progenitors present in bone marrow and fetal tissues where partial oxygen tension is low. Here we show that infected human primary erythroid progenitor cells exposed to hypoxia (1% O2) in vitro increase viral capsid protein synthesis, virus replication, and virus production. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor involved in the cellular response to reduced oxygenation, is shown to bind an HIF binding site (HBS) located in the distal part of the B19 promoter region, but the precise mechanism involved in the oxygen-sensitive upregulation of viral gene expression remains to be elucidated.

PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors.

BACKGROUND: Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs) with erythropoietin (Epo) leads to the activation of the mitogenic kinases (MEKs and Erks). How this is accomplished mechanistically remained unclear. RESULTS: Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K) family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors. CONCLUSIONS: These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis.

The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNA-binding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant TAL1 protein expression in human hematopoietic CD34(+) cells using a lentiviral vector. Differentiation capacities of the transduced cells were studied in a culture system that distinguishes early and late erythroid development. Results indicate that enforced TAL1 expression enhances long-term culture initiating cell (LTC-IC) potential and erythroid differentiation of human CD34(+) cells as shown by increased beta globin and porphobilinogen deaminase (PBGD) gene expressions and erythroid colony-forming units (CFU-Es), erythroid burst-forming units (BFU-Es), and glycophorin A-positive (GPA(+)) cell productions. Enforced expression of a TAL1 protein deleted of its DNA-binding domain (named Delta bTAL1) mimicked most TAL1 effects except for the LTC-IC enhancement, the down-regulation of the CD34 surface marker, and the GPA(+) cell production. These results provide the first functional indications of DNA-binding-dependent and -independent roles of TAL1 in human erythropoiesis.