Inhibition of the TGF-β receptor I kinase promotes hematopoiesis in MDS

Abstract MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of transforming growth factor–β (TGF-β) receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34+ cells, providing direct evidence of overactivation of TGF-β pathway in this disease. Suppression of the TGF-β signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-β–mediated gene activation in BM stromal cells, and reverses TGF-β–mediated cell-cycle arrest in BM CD34+ cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-β1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-β signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.

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Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis

Blood ◽

10.1182/blood-2010-11-318543 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2296-2304 ◽

Cited By ~ 52

Author(s):

Anupama Narla ◽

Shilpee Dutt ◽

J. Randall McAuley ◽

Fatima Al-Shahrour ◽

Slater Hurst ◽

...

Keyword(s):

Bone Marrow ◽

Expression Profiles ◽

Bone Marrow Failure ◽

Gene Expression Profiles ◽

Absolute Number ◽

Red Blood Cell Transfusion ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Cd34 Cells

Abstract Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.

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Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors

Blood ◽

10.1182/blood-2006-05-023093 ◽

2006 ◽

Vol 108 (13) ◽

pp. 4170-4177 ◽

Cited By ~ 86

Author(s):

Tony A. Navas ◽

Mani Mohindru ◽

Myka Estes ◽

Jing Ying Ma ◽

Lubomir Sokol ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

P38 Mapk ◽

Myelodysplastic Syndromes ◽

Clinical Investigation ◽

P38 Map Kinase ◽

Hematopoietic Progenitors ◽

Molecule Inhibitor ◽

Pharmacologic Inhibition

Abstract The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38α by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38α isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.

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Analysis of Leukemogenic Effects of RUNX1 and CSF3R Mutations Using Congenital Neutropenia (CN)/AML Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

Blood ◽

10.1182/blood.v128.22.404.404 ◽

2016 ◽

Vol 128 (22) ◽

pp. 404-404

Author(s):

Benjamin Dannenmann ◽

Maksim Klimiankou ◽

Christian Lindner ◽

Azadeh Zahabi ◽

Regine Bernhard ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Trisomy 21 ◽

Bone Marrow Failure ◽

Hematopoietic Cells ◽

Hematopoietic Progenitors ◽

Congenital Neutropenia ◽

Induced Pluripotent

Abstract Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome. Recently we reported a high frequency of cooperating RUNX1 and CSF3R mutations in CN patients that developed AML or MDS. Only a combination of these two mutations induced elevated proliferation and diminished myeloid differentiation of CD34+ cells in vitro. To confirm these clinical data in an in vitro model, we generated human induced pluripotent stem cells (hiPSCs) from PBMNCs of a CN patient harbouring p.C151Y mutation in ELANE after acquisition of AML. During GCSF treatment, this patient acquired G-CSFRmutation p.Q741*, which leads to a truncated G-CSF receptor and was detected six years prior to overt AML. Three years later, he acquired an additional RUNX1 (p.R139G) mutation, which is located in the RUNT-homology domain (RHD). Subsequently, he developed AML (FAB M1) with trisomy 21. Reprogramming of PBMNCs isolated from the time-point of AML (ca. 80 % of AML blasts) resulted in the generation of hiPSCs clones harbouring either only ELANE p.C151Y mutation (CN-iPSC clone, derived from non-leukemia PBMNCs) or additional CSF3R and RUNX1 mutations and trisomy 21 (CN/AML-iPSC clone, derived from AML blasts), which was subsequently validated by Sanger sequencing and by digital PCR. These iPSCs clones have been tested for their pluripotency and self-renewal capacity. Both iPSC clones expressed the pluripotent stem cell surface markers SSEA-4 and TRA-1-60 and displayed alkaline phosphatase activity. Further they highly expressed mRNA of the pluripotent stem cell markers SOX2, ABCG2, DNMT and NANOG and were able to differentiate into all three germ layers (meso-, endo- and ectoderm). Embryoid body (EB)-based hematopoietic / neutrophilic differentiation of CN-iPS clones using serum-free APEL stem cell differentiation medium showed comparable amounts of CD34+ and CD33+ cells, but ~ 2-fold reduction of CD16+ cells, compared to healthy donor (HD) iPSCs. CN/AML-iPSCs were not able to differentiate into mature granulocytes at all and revealed 10-fold reduced counts of CD34+ and CD33+hematopoietic cells. Morphological examinations of Giemsa-stained cytospin slides confirmed these results. Additionally, CN/AML-iPSCs showed a highly reduced number of CFU-G and CFU-GM colonies in CFU-Assay. To investigate the intracellular mechanisms of leukemogenic transformation in CN, we analyzed gene expression profiles of hematopoietic cells generated from CN-iPSCs vs CN/AML-iPSCs and HD-iPSCs for various time points of differentiation in our EB based-system. Our previous microarray-based analysis of bone marrow CD33+ cells of this CN/AML patient revealed that genes overexpressed in early hematopoietic stem/progenitor cells (HSPCs) as compared to more mature progenitors, such as DNTT, BAALC, CD34, HPGDS, NPR3 and PROM1 were strongly upregulated in CN/AML blasts harbouring both RUNX1 and CSF3R mutations, as compared to the cells prior to leukemia development. Intriguingly, elevated expression of these genes was described previously in RUNX1-mutated de novo AML blasts (Mendler et al., JCO 2012). This genetic signature suggests transformation of hematopoietic progenitors carrying mutated CSF3R into more primitive hematopoietic progenitors after aquisition of RUNX1mutation. We were able to confirm markedly increase of mRNA levels of these genes in hematopoietic cells derived from CN/AML-iPSCs, as compared to CN-iPSCs. In addition, we found that hematopoietic cells of both CN-iPSCs and CN/AML-iPSCs revealed increased expression of unfolded-protein response (UPR) genes DDIT3 (CHOP), ATF4 and ATF6, as compared to HD-iPSCs. Activation of UPR in hematopoietic cells of CN-ELANEpatients has been previously described by our and other groups. CN/AML-iPSC-derived hematopoietic progenitor cells expressed RUNX1 mRNA at least two-fold higher, as compared to HD- or CN-iPSC-derived cells. In summary, we established an in vitro cellular model of leukemogenic transformation in CN patients using CN/AML-patient derived hiPSCs that confirmed clinical data of Skokowa et al. (Blood 123:2550, 2014) on a cooperative leukemogenic effect of CSF3R and RUNX1 mutations. Comprehensive analysis of hematopoiesis using this iPSCs model will give us a deeper view into this highly complex signaling network operating during leukemogenic transformation of HSCs in pre-leukemic bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

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Interferon-γ-induced gene expression in CD34 cells: identification of pathologic cytokine-specific signature profiles

Blood ◽

10.1182/blood-2005-05-1884 ◽

2006 ◽

Vol 107 (1) ◽

pp. 167-175 ◽

Cited By ~ 78

Author(s):

Weihua Zeng ◽

Akira Miyazato ◽

Guibin Chen ◽

Sachiko Kajigaya ◽

Neal S. Young ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Expression Patterns ◽

Interferon Γ ◽

Molecular Signature ◽

Bone Marrow Failure Syndromes ◽

Cd34 Cells ◽

Ifn Γ

Abstract Hematopoietic effects of interferon-γ (IFN-γ) may be responsible for certain aspects of the pathology seen in bone marrow failure syndromes, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and some forms of myelodysplasia (MDS). Overexpression of and hematopoietic inhibition by IFN-γ has been observed in all of these conditions. In vitro, IFN-γ exhibits strong inhibitory effects on hematopoietic progenitor and stem cells. Previously, we have studied the transcriptome of CD34 cells derived from patients with bone marrow failure syndromes and identified characteristic molecular signatures common to some of these conditions. In this report, we have investigated genome-wide expression patterns after exposure of CD34 and bone marrow stroma cells derived from normal bone marrow to IFN-γ in vitro and have detected profound changes in the transcription profile. Some of these changes were concordant in both stroma and CD34 cells, whereas others were specific to CD34 cells. In general, our results were in agreement with the previously described function of IFN-γ in CD34 cells involving activation of apoptotic pathways and immune response genes. Comparison between the IFN-γ transcriptome in normal CD34 cells and changes previously detected in CD34 cells from AA and PNH patients reveals the presence of many similarities that may reflect molecular signature of in vivo IFN-γ exposure.

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Bone Marrow Failure in RPS19-Deficient Mice Is Partly Caused by p53 Activation and Responds to L-Leucine Treatment

Blood ◽

10.1182/blood.v118.21.727.727 ◽

2011 ◽

Vol 118 (21) ◽

pp. 727-727

Author(s):

Pekka Jaako ◽

Shubhranshu Debnath ◽

Karin Olsson ◽

Johan Flygare ◽

Stefan Karlsson

Keyword(s):

Bone Marrow ◽

Preliminary Data ◽

Bone Marrow Failure ◽

Mtor Pathway ◽

Wild Type ◽

Hematopoietic Progenitors ◽

P53 Activation ◽

Deficient Mice

Abstract Abstract 727 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al. Blood. 2010;116:193. ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. In the current study we have analyzed the role of p53 in RPS19-deficient hematopoiesis by crossing the transgenic mice into Trp53 null background. To isolate the hematopoietic phenotype we transplanted bone marrow cells from these mice into lethally irradiated wild-type recipients. We have previously shown that the severity of the hematopoietic phenotype in transplanted recipients is highly dependent on the level of RPS19 downregulation, and the recipients with low RPS19 expression die 2–3 weeks after induction because of bone marrow failure. Remarkably, the inactivation of Trp53 rescued the early mortality in these recipients. However, although the inactivation of Trp53 completely reversed the erythrocyte and leukocyte numbers in the recipients with intermediate RPS19 downregulation, the recipients with low RPS19 expression still developed a mild anemia and macrocytosis. p53 activation is known to inhibit the AKT/mTOR pathway, a central regulator of cell growth and survival. Although the role of this pathway in DBA pathogenesis remains poorly defined, some patients positively respond to treatment with amino acid L-leucine, a nutrient signal that stimulates mTOR activity. Currently we are studying the role of L-leucine in RPS19-deficient hematopoiesis both in vitro and in vivo. Our preliminary data confirm that L-leucine modestly enhances the proliferation of RPS19-deficient c-Kit -enriched hematopoietic progenitors in vitro (1.2 fold in 8 days), while there is no effect on wild-type cultures. Interestingly, the proliferative response in RPS19-deficient cultures appears more pronounced when cells are cultured in low cytokine concentration (1.6 fold in 8 days). Since primary cells from DBA patients are highly responsive to stem cell factor (SCF), which also mediates its effect partly via PI3K/AKT/mTOR pathway, we are studying whether L-leucine has a synergistic role with SCF enhancing the proliferation of hematopoietic progenitors. Finally, a 15% L-leucine supplement in drinking water partly rescues the erythrocyte and leukocyte number in RPS19-deficient mice. In summary, our results demonstrate a key role of p53 activation in RPS19-deficient DBA, although they also suggest that p53-independent pathways may contribute towards phenotype upon severe RPS19 deficiency. Furthermore, our preliminary data supports the role of L-leucine as a therapeutic agent in the treatment of DBA. Disclosures: No relevant conflicts of interest to declare.

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Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽

10.1182/blood.v118.21.sci-2.sci-2 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-2-SCI-2

Author(s):

Stefan Karlsson ◽

Johan Flygare ◽

Pekka Jaako ◽

David Bryder

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

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TGF-β Pathway Inhibition Rescues the Function of Hematopoietic Stem and Progenitor Cells Derived from Patients with Fanconi Anemia

Blood ◽

10.1182/blood.v126.23.297.297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 297-297

Author(s):

Haojian Zhang ◽

David Kozono ◽

Kevin O'Connor ◽

Alix Rousseau ◽

Lisa Moreau ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Neutralizing Antibody ◽

Genotoxic Stress ◽

Cross Linking ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Pathway Inhibition

Abstract Introduction: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients develop bone marrow failure during the first decade of life, and frequently require an allogeneic or unrelated donor bone marrow transplant. FA patients also develop other hematologic manifestations, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) due to clonal evolution. FA is caused by biallelic mutation in one of eighteen FANC genes, the products of which cooperate in the FA/BRCA DNA repair pathway and regulate cellular resistance to DNA cross-linking agents. Bone marrow failure in FA is attributable to an impaired hematopoietic stem and progenitor cell (HSPC) pool. HSPCs in FA patients and FA mice exhibit reduced cell number and compromised stem cell function. Recent studies suggest that bone marrow failure in FA and impaired HSPC function result from the genotoxicity of endogenous cross-linking agents or from physiological stress. A greater understanding of the mechanisms of impairment of HSPC function could improve the therapeutic options for FA patients. Using a whole genome-wide shRNA screen, we have recently identified that the canonical transforming growth factor-β (TGF-β) pathway plays an important growth suppressive role in FA and targeting this pathway can reduce the genotoxic stress-induced growth inhibition of FA cells. Here, we investigated the possible suppressive function of the TGF-β pathway in HSPCs derived from patients with FA. Methods: We performed in vitro colony-forming assays using primary FA patient- derived bone marrow CD34+ cells which were either transduced with shRNA targeting SMAD3 or treated with the anti-human TGF-β neutralizing antibody GC1008. FA-like HSPCs were generated by stably knocking down FANCD2 with lentivirus encoded shRNA in primary human cord blood CD34+ cells. An in vivo engraftment assay was performed by transplanting the FA-like HSPCs into irradiated NSG mice. Results: The primary human FA bone marrow cells displayed elevated mRNA expression of multiple TGF-β pathway components. The TGF-β pathway inhibition, by knockdown of SMAD3 or anti-human TGF-β neutralizing antibody GC1008, rescued the in vitro clonogenic defects of primary CD34+ cells from bone marrow of five different FA patients. Similarly, the TGF-β pathway disruption by depletion of SMAD3 or GC1008 antibody in primary FA-like HSPCs, also rescued their clonogenic defect, and partially restored genotoxic stress-induced growth inhibition. Further, as the very low number of CD34+ cells in FA patients did not allow efficient xenograft assay to analyze in vivo clonogenicity, we performed a surrogate in vivo xenograft assay using FA-like primary CD34+ cells. Importantly, blockade of the TGF-β pathway by GC1008 antibody treatment enhanced the engraftment potential of primary FA-like CD34+ cells in vivo. Collectively, these results demonstrated that increased TGF-β pathway signaling impairs the hematopoietic function of primary human FA HSPCs. Conclusions: The TGF-β pathway signaling is increased in primary FA patient-derived hematopoietic cells and blockade of this pathway can restore the function of human FA-deficient primary HSPCs. The TGF-β signaling pathway-mediated growth suppression may account, at least in part, for bone marrow failure in FA. This work suggests that the TGF-β signaling pathway provides a novel therapeutic target for the treatment of bone marrow failure in FA. Disclosures No relevant conflicts of interest to declare.

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Transforming Growth Factor-β1 (TGF-β1) Induces Thrombopoietin From Bone Marrow Stromal Cells, Which Stimulates the Expression of TGF-β Receptor on Megakaryocytes and, in Turn, Renders Them Susceptible to Suppression by TGF-β Itself With High Specificity

Blood ◽

10.1182/blood.v94.6.1961 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1961-1970 ◽

Cited By ~ 61

Author(s):

Sumio Sakamaki ◽

Yasuo Hirayama ◽

Takuya Matsunaga ◽

Hiroyuki Kuroda ◽

Toshiro Kusakabe ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Transforming Growth Factor ◽

High Specificity ◽

Transforming Growth Factor Β1 ◽

Lineage Specificity ◽

Β Receptor ◽

Tgf Β1

Abstract The present study was designed to test the concept that platelets release a humoral factor that plays a regulatory role in megakaryopoiesis. The results showed that, among various hematoregulatory cytokines examined, transforming growth factor-β1 (TGF-β1) was by far the most potent enhancer of mRNA expression of bone marrow stromal thrombopoietin (TPO), a commitment of lineage specificity. The TPO, in turn, induced TGF-β receptors I and II on megakaryoblasts at the midmegakaryopoietic stage; at this stage, TGF-β1 was able to arrest the maturation of megakaryocyte colony-forming units (CFU-Meg). This effect was relatively specific when compared with its effect on burst-forming unit-erythroid (BFU-E) or colony-forming unit–granulocyte-macrophage (CFU-GM). In patients with idiopathic thrombocytopenic purpura (ITP), the levels of both TGF-β1 and stromal TPO mRNA were correlatively increased and an arrest of megakaryocyte maturation was observed. These in vivo findings are in accord with the aforementioned in vitro results. Thus, the results of the present investigation suggest that TGF-β1 is one of the pathophysiological feedback regulators of megakaryopoiesis.

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In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice

Blood ◽

10.1182/blood.v100.6.2032 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2032-2039 ◽

Cited By ~ 42

Author(s):

Paula Rı́o ◽

José Carlos Segovia ◽

Helmut Hanenberg ◽

JoséAntonio Casado ◽

Jesús Martı́nez ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Fanconi Anemia ◽

Fluorescent Protein ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Retroviral Vectors ◽

Growth Defect ◽

Hematopoietic Progenitors

Abstract Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca−/− mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect ofFanca−/−progenitors, either when total BM or when purified Lin−Sca-1+ cells were subjected to in vitro stimulation. Liquid cultures ofFanca−/−BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca−/−progenitors, purified Lin−Sca-1+ cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCAgenes. Lin−Sca-1+ cells fromFanca−/−mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors ofFanca−/−mice.

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In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice

Blood ◽

10.1182/blood.v100.6.2032.h81802002032_2032_2039 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2032-2039 ◽

Cited By ~ 2

Author(s):

Paula Rı́o ◽

José Carlos Segovia ◽

Helmut Hanenberg ◽

JoséAntonio Casado ◽

Jesús Martı́nez ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Fanconi Anemia ◽

Fluorescent Protein ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Retroviral Vectors ◽

Growth Defect ◽

Hematopoietic Progenitors

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca−/− mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect ofFanca−/−progenitors, either when total BM or when purified Lin−Sca-1+ cells were subjected to in vitro stimulation. Liquid cultures ofFanca−/−BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca−/−progenitors, purified Lin−Sca-1+ cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCAgenes. Lin−Sca-1+ cells fromFanca−/−mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors ofFanca−/−mice.

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