Hematopoietic Progenitor Cell Development Research Articles

WNT9A Is a Conserved Regulator of Hematopoietic Stem and Progenitor Cell Development.

Hematopoietic stem cells (HSCs) differentiate into all cell types of the blood and can be used therapeutically to treat hematopoietic cancers and disorders. Despite decades of research, it is not yet possible to derive therapy-grade HSCs from pluripotent precursors. Analysis of HSC development in model organisms has identified some of the molecular cues that are necessary to instruct hematopoiesis in vivo, including Wnt9A, which is required during an early time window in zebrafish development. Although bona fide HSCs cannot be derived in vitro, it is possible to model human hematopoietic progenitor development by differentiating human pluripotent stem cells to hematopoietic cells. Herein, we modulate WNT9A expression during the in vitro differentiation of human embryonic stem cells to hematopoietic progenitor cells and demonstrate that WNT9A also regulates human hematopoietic progenitor cell development in vitro. Overexpression of WNT9A only impacts differentiation to CD34+/CD45+ cells during early time windows and does so in a dose-dependent manner. The cells that receive the Wnt signal—not the cells that secrete WNT9A—differentiate most efficiently to hematopoietic progenitors; this mimics the paracrine action of Wnt9a during in vivo hematopoiesis. Taken together, these data indicate that WNT9A is a conserved regulator of zebrafish and human hematopoietic development.

Flt3 Ligand Treatment Attenuates T Cell Dysfunction and Improves Survival in a Murine Model of Burn Wound Sepsis.

Sepsis is a leading cause of death among severely burned patients. Burn injury disrupts the protective skin barrier and causes immunological dysfunction. In our previous studies, we found that burn injury and wound infection causes a significant decline in lymphocyte populations, implying adaptive immune system dysfunction. In the present study, we examined the effect of treatment with Fms-like tyrosine kinase-3 Ligand (Flt3L) on T cell phenotype and function in a model of burn wound sepsis. FLt3L is an essential cytokine required for hematopoietic progenitor cell development and expansion of both myeloid and lymphoid lineages. Flt3L has been shown to potentiate innate immune functions of dendritic cells and neutrophils during burn wound sepsis. However, the ability of Flt3L to improve T cell function during burn wound sepsis has not been previously evaluated. Mice underwent 35% total body surface area scald burn and were treated with Flt3L (10 μg) or vehicle daily via the intraperitoneal route starting 1 day after burn injury. On day 4 after burn injury, Pseudomonas aeruginosa was used to induce wound infection. Leukocytes in spleen and wound draining lymph nodes were characterized using flow cytometry. Bacterial clearance, organ injury, and survival were also assessed. Flt3L treatment prevented the decline in splenic CD4 and CD8 T cells caused by burn injury and infection. Flt3L treatment also attenuated the decline in CD28 expression on CD4 and CD8 T cells and IFNγ production by CD8 T cells in the spleen and wound draining lymph nodes. Furthermore, Flt3L decreased the levels of programmed death ligand 1 expression on splenic dendritic cells and macrophages. Flt3 treatment improved systemic bacterial clearance, decreased liver and kidney injury, and significantly improved survival in mice with burn wound sepsis. Burn injury and associated sepsis causes significant loss of T cells and evidence of T cell dysfunction. Flt3L attenuates T cell dysfunction and improves host resistance to burn wound sepsis in mice.

Use of Human Pluripotent Stem Cells to Model the Hematopoietic Defect and Repair of Human Telomerase Deficiency

Telomeres are repetitive DNA protein structures that cap the ends of chromosomes, protect chromosome ends from degradation and fusion, and are essential for maintenance of genomic integrity. Telomere length has been shown to gradually shorten over time as cells divide. When telomeres become critically short, the cells enter a state of senescence. As such, telomere shortening has been implicated in accelerated aging. Defects in telomerase function have been associated with the development of bone marrow failure. Patients with inherited mutations in telomerase components have significantly shortened telomere lengths and reductions in telomere length have been associated with a worse prognosis in myelodysplastic syndrome (MDS) and Aplastic anemia (AA). We have isolated fibroblasts from patients with a novel mutation in the telomerase RNA component (TERC), a 6 nucleotide in frame duplication at position +334, which results in bone marrow failure. We have derived several lines of human induced pluripotent stem cells (iPSCs) from these patients. We have demonstrated that these telomerase-deficient iPSCs appropriately express markers of pluripotency: Oct4, Sox2, SSEA4, and Nanog. Using quantitative real time PCR, we were able to measure the average telomere length as a T/S ratio of kilobases of telomere length per genome. We have determined that reprogramming results in significant increase in telomere length of control fibroblasts. However despite the typical induction of endogenous TERT expression during reprograming in our telomerase-deficient iPSCs, these TERC-mutant iPSCs did not demonstrate significant telomere elongation. We found telomerase-deficient fibroblasts have telomere lengths of 130-150kb/diploid genome compared to normal human fibroblasts with telomeres of ~250kb/diploid genome. After reprogramming, the iPSCs generated from wild type fibroblasts can have markedly increased telomere lengths to as high as 2000kb/diploid genome. The telomere lengths in TERC-mutant iPSCs from two different patients are less than 300kb/diploid genome (replicates=3). Expression of telomerase components TERT and TERC, DKC was compared to the mRNA level by qRT-PCR in the TERC-mutant iPSCs. We found significant variation in mRNA expression levels of telomerase components the different lines of telomerase deficient iPSCs, and even variation among different clones of the same telomerase deficient line. We have also found that mutations in TERC results in defective hematopoietic differentiation from these iPSCs in in vitro assays. In the TERC-mutant iPSCs, the proportion of CD34+CD45+ hematopoietic cells was reduced compared to wild type controls. Wild type iPSCs produce 30% CD34+ cells compared to 15-20% in TERC-mutant iPSCs (n=3). Additionally, wild type iPSC controls produce 9-10% CD34+CD45+ hematopoietic progenitor cells compared to 1-2% of TERC mutant cells. Interestingly, approximately equal proportions (8-9%) of wild type and TERC-mutant cells differentiate into CD34+CD31+ endothelial cells, suggesting this pathway is less affected by the TERC mutation. The TERC-mutant iPSCs demonstrate reduced development of hematopoietic progenitor cells in standard hematopoietic colony forming cell assays: 100 CFCs per 50,000 differentiated wild type iPSC-derived cells compared to 65 CFCs per 50,000 differentiated TERC mutant iPSC-derived cells. In order to rescue the hematopoietic defect in telomerase deficient TERC-mutant iPSCs we have used the Sleeping Beauty transposon system to over express these telomerase components TERC and TERT in the TERC mutant iPSCs. We are currently characterizing the effect of overexpression of these telomerase components on hematopoietic differentiation to determine if this provides a strategy to enable use of gene-corrected iPSCs to provide a future therapy for patients with bone marrow failure due to defined telomerase deficiencies. DisclosuresNo relevant conflicts of interest to declare.

Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish.

Hemangioblast, including primitive hematopoietic progenitor cells, play an important role in hematopoietic development, however, the underlying mechanism for the propagation of hematopoietic progenitor cells remains elusive. A variety of regulatory molecules activated in early embryonic development play a critical role in the maintenance of function of hematopoietic progenitor cells. Homeobox transcription factors are an important class of early embryonic developmental regulators determining hematopoietic development. However, the effect of homeobox protein Hox-B4 (HOXB4) ectopic expression on the development of hemangioblasts has not been fully addressed. This study aimed to investigate the role of Hoxb4a, an ortholog gene of HOXB4 in zebrafish, in the hematopoietic development in zebrafish. A transgenic zebrafish line was established with Cre-loxP system that stably overexpressed enhanced green fluorescent protein (EGFP)-tagged Hoxb4a protein under the control of hemangioblast-specific lmo2 promoter. Overexpression of Hoxb4a in the development of hemangioblasts resulted in a considerable increase in the number of stem cell leukemia (scl) and lmo2-positive primitive hematopoietic progenitor cells occurring in the posterior intermediate cell mass (ICM). Interestingly, Hoxb4a overexpression also disrupted the development of myelomonocytes in the anterior yolk sac and the posterior ICM, without affecting erythropoiesis in the posterior ICM. Taken together, these results indicate that Hoxb4a favours the development of hematopoietic progenitor cells originated from hemangioblasts in vivo.

Gfi-1 regulates the erythroid transcription factor network through Id2 repression in murine hematopoietic progenitor cells

AbstractGrowth factor independence 1 (Gfi-1) is a part of the transcriptional network that regulates the development of adult hematopoietic stem and progenitor cells. Gfi-1-null (Gfi-1−/−) mice have reduced numbers of hematopoietic stem cells (HSCs), impaired radioprotective function of hematopoietic progenitor cells (HPCs), and myeloid and erythroid hyperplasia. We found that the development of HPCs and erythropoiesis, but not HSC function, was rescued by reducing the expression of inhibitor of DNA-binding protein 2 (Id2) in Gfi-1−/− mice. Analysis of Gfi-1−/−;Id2+/− mice revealed that short-term HSCs, common myeloid progenitors (CMPs), erythroid burst-forming units, colony-forming units in spleen, and more differentiated red cells were partially restored by reducing Id2 levels in Gfi-1−/− mice. Moreover, short-term reconstituting cells, and, to a greater extent, CMP and megakaryocyte-erythroid progenitor development, and red blood cell production (anemia) were rescued in mice transplanted with Gfi-1−/−;Id2+/− bone marrow cells (BMCs) in comparison with Gfi-1−/− BMCs. Reduction of Id2 expression in Gfi-1−/− mice increased the expression of Gata1, Eklf, and EpoR, which are required for proper erythropoiesis. Reducing the levels of other Id family members (Id1 and Id3) in Gfi-1−/− mice did not rescue impaired HPC function or erythropoiesis. These data provide new evidence that Gfi-1 is linked to the erythroid gene regulatory network by repressing Id2 expression.

Go6976, a Potent FLT3 Kinase Inhibitor, Exerts Antiproliferative Activity Against Acute Myeloid Leukemia Via Inhibition Of Survivin and Mcl-1 In The Presence Of Human Serum

The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis, and thus recent studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. Go6976 is an indolocarbazole with a simillar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. Its IC50 value is 0.7nM. We also tested the effect of Go6976 on several kinds of other kinases. Go6976 inhibited the kinase activity of Aurora-A, Aurora-B and JAK2 with IC50 values of 118.2 nM, 77.7 nM and 92.7 nM, respectively. Go6976 did not show the inhibitory activity against the FGFR3 even at 1 microM. These data indicated Go6976 preferentially and potently inhibit the FLT3. Go6976 significantly inhibited the proliferation of human leukemia cells having FLT3-ITD. The IC50 values of Go6976 against MV4-11 and MOLM-13 were 0.044 and 0.008 microM, respectively. In contrast, human leukemia HL-60 and U937 cells which lack FLT3-ITD showed strong resistance to Go6976 treatment. Furthermore, we observed that Go6976 shows minimal toxicity for purified human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 in MV4-11 and MOLM13 cells. Consistent with FLT3 inhibition, Go6976 potently inhibited phosphorylation of constitutively activated STAT3/5, Erk1/2, and Akt. Western blotting analysis revealed that MV4-11 and MOLM13 cells possess abundant survivin and Mcl-1 protein. We hypothesized that the expression of survivin and Mcl-1 may be regulated by constitutive activation of FLT3. In order to test this hypothesis, the effect of siRNA for FLT3-ITD was examined. Indeed, we observed that siRNA-induced down-regulation of FLT3 decreased survivin and Mcl-1 expression in MOLM13 cells, suggesting that up-regulated survivin and Mcl-1 may be closely associated with FLT3 signaling. Interestingly, we found that both survivin mRNA and protein were rapidly downregulated by Go6976 treatment in MOLM13 and MV4-11 cells. It was also observed that Go6976 significantly suppressed Mcl-1 mRNA and protein. It has been reported that STAT-3 and STAT-5 signaling play a pivotal role in the regulation of survivin and Mcl-1, respectively. Thus, inhibitory effects of Go6976 on the expression Survivin and Mcl-1 may be a consequence of the suppression of phosphorylation of STAT-3 and STAT-5 by Go6976 in FLT3-ITD cells. This inhibition of anti-apoptotic proteins by Go6976 may be critical for its antiproliferative effect in FLT3-ITD cells. It has been known that previous FLT3 inhibitors such as PKC412 and CEP-701 bind to the human plasma protein, alpha1-acid glycoprotein, resulting in significantly diminished inhibitory activity against FLT3. Indeed, inhibitory effect of PKC412 on FLT3 was significantly decreased, when MOLM13 cells were treated with PKC412 in the presence of human serum. In contrast, we found that Go6976 potently inhibits phosphorylation of FLT3 and exerts cytotoxicity even in the presence of human serum or human alpha1-acid glycoprotein. In conclusion, our data indicate that Go6976 may have a unique therapeutic potential for FLT3-driven acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

A Simple Model System Enabling Human CD34+ Cells to Undertake Differentiation Towards T Cells

BackgroundChannelling the development of haematopoietic progenitor cells into T lymphocytes is dependent upon a series of extrinsic prompts whose temporal and spatial sequence is critical for a productive outcome. Simple models of human progenitor cells development depend in the main on the use of xenogeneic systems which may provide some limitations to development.Methods and FindingsHere we provide evidence that a simple model system which utilises both human keratinocyte and fibroblast cell lines arrayed on a synthetic tantalum coated matrix provides a permissive environment for the development of human CD34⁺ haematopoietic cells into mature CD4⁺ or CD8⁺ T lymphocytes in the presence of Interleukin 7 (IL-7), Interleukin 15 (IL-15) and the Fms-like tyrosine kinase 3 ligand (Flt-3L). This system was used to compare the ability of CD34+ cells to produce mature thymocytes and showed that whilst these cells derived from cord blood were able to productively differentiate into thymocytes the system was not permissive for the development of CD34+ cells from adult peripheral blood.Conclusions/SignificanceOur study provides direct evidence for the capacity of human cord blood CD34+ cells to differentiate along the T lineage in a simple human model system. Productive commitment of the CD34⁺ cells to generate T cells was found to be dependent on a three-dimensional matrix which induced the up-regulation of the Notch delta-like ligand 4 (Dll-4) by epithelial cells.

Abstract 1806: The indolocarbazole derivative Go6976 suppresses the growth of leukemia cells with FLT3 mutations

Abstract The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML), and inhibitors of FLT3 are of clinical interest. Go6976 is an indolocarbazole inhibitor of the calcium-dependent isozymes of protein kinase C (PKC). In the present study, we demonstrated that Go6976 possesses a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. We also showed that Go6976 is a potent inhibitor of Aurora-B kinase. Go6976 significantly inhibited proliferation of leukemia MV4-11 cells having FLT3-internal tandem duplication (ITD). Cell growth inhibition by Go6976 occurred in parallel with the drug inhibiting FLT3 and its downstream signal pathways such as extracellular signal-regulated kinase1/2, STAT-5 and p38. The FLT3-independent myeloid leukemia HL-60 and U937 showed strong resistance to Go6976 treatment. Induction of massive apoptosis were observed upon treatment with Go6976 in MV4-11 cells associated with significant down-regulation with Survivin and Mcl-1 protein. Interestingly, Go6976 treatment also inhibited Survivin phosphorylation on Thr34, which is critical for its anti-apoptotic activity. This inhibition of Survivin phosphorylation occurred due to the direct suppression of Aurora-B by Go6976 treatment. These data indicate that Go6976 may have a unique therapeutic potential for patients with FLT3-driven leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1806. doi:1538-7445.AM2012-1806

Abstract 5217: RNA editing enzyme ADAR1 drives leukemia stem cell differentiation and self-renewal in chronic myeloid leukemia

Abstract Chronic myeloid leukemia (CML) is the first cancer that was shown to originate from a genetic abnormality - the Philadelphia chromosome translocation, and production of its constitutively active protein tyrosine kinase product, BCR-ABL. The disease progresses slowly from chronic phase to accelerated phase, and later transforms to blast crisis (BC) stage. Cancer stem cells (CSCs) are a subset of tumor cells that have acquired certain treatment-resistant stem cell properties. High levels of RNA editing are associated with a primitive transcriptional program typical of human embryonic stem cells, and RNA editing plays an important role in both embryonic hematopoietic cell fate determination and in maintenance of normal hematopoiesis. Human RNA editing occurs primarily in secondary structures created by Alu retroelements and is carried out by enzymes such as the adenosine deaminase acting on RNA (ADAR) family. Among these, ADAR1 was also recently shown to be required for normal hematopoiesis by suppressing interferon-induced apoptosis. Our research focuses on dissecting the role of ADAR-mediated RNA editing in normal human hematopoietic progenitor cell development compared with malignant editing programs that may be activated in leukemia stem cells (LSC) during the progression of human CML. Our data demonstrates that BC LSC harbor increased levels of the interferon-responsive ADAR1 p150 isoform compared with chronic phase (CP) progenitors and normal cord blood progenitors. Expression of this isoform also exhibits a positive correlation with BCR-ABL expression levels - an effect which is specific to BC progenitors, suggesting that ADAR1 expression correlates with disease progression from CP to BC. In vitro hematopoietic progenitor assays with normal cord blood progenitors and CP samples transduced with lentiviral vectors overexpressing human ADAR1 reveals a significant shift in cell differentiation fate towards granulocyte-macrophage progenitor (GMP) colonies, which has been shown to be the initiating LSC population in CML Correspondingly, a progression towards erythroid lineage was observed in BC CML LSC transduced with lentiviral vectors expressing shRNA targeting ADAR1. Further qRT-PCR analyses revealed that the mechanism through which ADAR1 drives LSC and HSC differentiation towards myelopoiesis involves regulation of PU.1, which in turn inhibits GATA1 expression. Moreover, in vivo studies in a robust humanized CML mouse model showed a significant decrease in LSC serial transplantation potential of lentiviral shADAR1-transduced BC progenitors transplanted into neonatal RAG2-/-γc-/- mice. Together, these data support a crucial role for ADAR1 in cell fate determination and self-renewal potential of hematopoietic stem cells in both normal human progenitors and in malignant LSC that drive disease progression and therapeutic resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5217. doi:1538-7445.AM2012-5217

Patterns of Molecular Response to and Relapse After Combination of Sorafenib, Idarubicin, and Cytarabine in Patients With FLT3 Mutant Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML), and inhibitors of FLT3 are of clinical interest. Sorafenib is an orally active multikinase inhibitor with potent activity against FLT3 and the Raf/ERK/MEK kinase pathway. We studied the patterns of molecular response and relapse in 18 patients with mutated FLT3 treated with the combination of sorafenib, idarubicin, and cytarabine. The median follow-up was 9 months. Sixteen patients achieved complete remission (CR), and the other 2 patients achieved CR but lacked platelet recovery for an overall response rate of 100%. Ten patients had their FLT3-mutated clone eradicated, with 6 patients who showed some residual FLT3-mutated cells, and 2 patients who showed persistent FLT3-mutated cells. The elimination of FLT3-mutated population at the time of morphologic CR, however, was not predictive of relapse. After a median follow-up of 9 months (range, 1-16 months), 10 (55%) patients had relapsed, with a median CR duration of 8.8 months (range, 1-9.5 months). By DNA sequencing, there was no evidence of an acquired FLT3 point mutation at the time of relapse in 7 patients tested, which suggested the presence of other mechanisms of sorafenib resistance. Sorafenib, combined with chemotherapy, is effective in attaining CR, but relapses still occur.

1 A mouse model highlights a role for p53 in 5q − syndrome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

The role of the GPR91 ligand succinate in hematopoiesis

Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development.

HIV-1 Infection of Bone Marrow Hematopoietic Progenitor Cells and Their Role in Trafficking and Viral Dissemination

Patients with HIV-1 often present with a wide range of hematopoietic abnormalities, some of which may be due to the presence of opportunistic infections and to therapeutic drug treatments. However, many of these abnormalities are directly related to HIV-1 replication in the bone marrow (BM). Although the most primitive hematopoietic progenitor cells (HPCs) are resistant to HIV-1 infection, once these cells begin to differentiate and become committed HPCs they become increasingly susceptible to HIV-1 infection and permissive to viral gene expression and infectious virus production. Trafficking of BM-derived HIV-1-infected monocytes has been shown to be involved in the dissemination of HIV-1 into the central nervous system (CNS), and it is possible that HIV-1 replication in the BM and infection of BM HPCs may be involved in the early steps leading to the development of HIV-1-associated dementia (HAD) as an end result of this cellular trafficking process. In addition, the growth and development of HPCs in the BM of patients with HIV-1 has also been shown to be impaired due to the presence of HIV-1 proteins and changes in the cytokine milieu, potentially leading to an altered maturation process and to increased cell death within one or more BM cell lineages. Changes in the growth and differentiation process of HPCs may be involved in the generation of monocyte populations that are more susceptible and/or permissive to HIV-1, and have potentially altered trafficking profiles to several organs, including the CNS. A monocyte subpopulation with these features has been shown to expand during the course of HIV-1 disease, particularly in HAD patients, and is characterized by low CD14 expression and the presence of cell surface CD16.

Hematopoietic Differentiation of Human Embryonic Stem Cells by Cocultivation with Stromal Layers

Human embryonic stem (hES) cells are of remarkable interest both for the utility of these cells for studying basic human developmental biology and as a potential source for novel therapeutics. Here, we provide detailed methodologies of one of the first systems used to mediate differentiation of hES cells--stromal cell coculture. Use of stromal cells adds the ability to manipulate aspects of the developmental niche that support differentiation into a defined lineage. These methods will allow efficient and reproducible development of hematopoietic progenitor cells, as well as potentially mature hematopoietic cells that are suitable for subsequent in vitro and in vivo studies.

Isolation and development of haematopoietic progenitor cells from peripheral blood of adult and newborn pigs

Pluripotent stem cells (PSCs), already described in human beings, are fibroblast-like cells that exhibit a CD34 marker specific for haematopoietic stem cells. In this work we have demonstrated the presence of PSCs in the peripheral blood of pigs, a species frequently used in transplantation studies as an animal model for human diseases. Differentiation into haematopoietic colonies (granulomacrophagic colonies, erythroid colonies and mixed colonies) has been carried out with the peripheral blood of adult and newborn pigs, using solely human commercial media. Peripheral blood mononuclear cells (PBMNCs) were cultured in semisolid methylcellulose based media enriched with recombinant human cytokines, achieving granulomacrophagic-colony forming unit (GM-CFU) and mixed-colony forming unit (Mix-CFU) growth with erythroblastic lineage proliferation in the presence of erythropoietin (Epo). In all the samples CFU growth was associated with the presence of recombinant human cytokine. No evidence of proliferation in control plates without cytokines was found. From liquid medium culture, a population of macrophages and CD34+ fibroblast like cells were retrieved 21 days after sowing. These findings allow us to think about the direct application of this simple and standardised method in several work fields such as the study of pharmacological effects of many drugs over the haematopoietic line and in the study of new strategies in cellular therapy for some human diseases.

A critical function for B-Raf at multiple stages of myelopoiesis

AbstractRaf kinases play an integral role in the classic mitogen-activated protein (MA3332) kinase (Raf/MEK/extracellular signal-related kinase [ERK]) intracellular signaling cascade, but their role in specific developmental processes is largely unknown. Using a genetic approach, we have identified a role for B-Raf during hematopoietic progenitor cell development and during megakaryocytopoiesis. Fetal liver and in vitro embryonic stem (ES) cell–derived myeloid progenitor development is quantitatively impaired in the absence of B-Raf. Biochemical data suggest that this phenotype is due to the loss of a normally occurring rise in B-Raf expression and associated ERK1/2 activation during hematopoietic progenitor cell formation. However, the presence of B-raf–/– ES cell–derived myeloid progenitors in the bone marrow of adult chimeric mice indicates the lack of an obligate cell-autonomous requirement for B-Raf in myeloid progenitor development. The lack of B-Raf also impairs megakaryocytopoiesis. Thrombopoietin (Tpo)–induced in vitro expansion of ES cell–derived megakaryocyte-lineage cells fails to occur in the absence of B-Raf. Moreover, this quantitative in vitro defect in megakaryocyte-lineage expansion is mirrored by chimeric mice data that show reduced B-raf–/– genotype contribution in megakaryocytes relative to its contribution in myeloid progenitors. Together, these data suggest that B-Raf plays a cell-autonomous role in megakaryocytopoiesis and a permissive role in myeloid progenitor development.

NF-κB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.

Molecular Isolation and Characterization of a Soluble Isoform of Activated Leukocyte Cell Adhesion Molecule That Modulates Endothelial Cell Function

Cell adhesion molecules regulate a variety of endothelial cell functions such as migration, response to inflammation, and angiogenesis. Recently, activated leukocyte cell adhesion molecule (ALCAM), a member of the Ig superfamily, has been detected in the primitive subsets of hematopoietic cells and endothelial cells during embryogenesis. ALCAM supports the development of hematopoietic cells as well as enhancing capillary tube formation in vitro. Here, we isolated a novel soluble isoform of ALCAM (sALCAM) that is produced via alternative splicing. sALCAM contains the single amino-terminal Ig-like domain of ALCAM and lacks a transmembrane domain. When expressed in cultured cells, sALCAM was properly secreted into the media. Both ALCAM and sALCAM are expressed in a variety of cultured human endothelial cells. Notably, their transcripts were differentially regulated in human microvascular endothelial cells (HMVEC) upon tumor necrosis factor-alpha stimulation. ALCAM significantly enhanced tube formation of endothelial-like yolk sac cells on Matrigel, whereas it inhibited their migration in vitro. sALCAM completely abolished these effects of ALCAM. Furthermore, sALCAM enhanced migration of mock-transfected endothelial-like yolk sac cells that do not express ALCAM, indicating that sALCAM has an independent effect on cell migration in addition to modulating ALCAM function. In addition, sALCAM significantly enhanced migration of HMVEC, whereas it inhibited tube formation of HMVEC on Matrigel. sALCAM demonstrated an ability to bind ALCAM and partially inhibited ALCAM-ALCAM homophilic interactions. Taken together, these data characterize a novel soluble isoform of ALCAM that may have ALCAM-dependent and ALCAM-independent functions, providing further insights regarding the role of this adhesion molecule in the regulation of endothelial cell function.

ALCAM (CD166): its role in hematopoietic and endothelial development

A critical role for the endothelium of yolk sac and dorsal aorta has been shown in embryonic hematopoiesis. A stromal cell line derived from yolk sac, YSCL-72, has been chosen to search for a novel molecule associated with embryonic hematopoiesis. Analysis between YSCL-72 and an adult aorta-derived endothelial cell line, EOMA, demonstrated that activated leukocyte cell adhesion molecule (ALCAM, or CD166) was specifically expressed in YSCL-72 but not in EOMA. Immunohistochemical study showed that ALCAM was expressed in the endothelium of yolk sac and dorsal aorta but not in adult aorta. ALCAM-transfected EOMA cells supported development of hematopoietic progenitor cells compared with vector-transfected EOMA cells, suggesting that ALCAM appeared to be crucial for hematopoiesis. In addition, ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation. Taken together with these findings, ALCAM is highly associated not only with embryonic hematopoiesis but also vasculoangiogenesis.