Exhaustion in Myeloid Lineage and Very Early Defect in HSPC Pool: An Embryonic Origin of Fanconi Haematological Disorders

Abstract Fanconi anemia (FA) is a genetic disorder due to mutations in one of the sixteen FANC genes involved in DNA repair. Many FA patients develop bone marrow failure (BMF) during childhood, and FA strongly predisposes to myelodysplasia syndrome and/or acute myeloid leukaemia. The pathogenesis of the BMF remains uncompletely understood. Low hematopoietic progenitor cell (HPCs) counts observed early in life and preceeding the onset of blood cytopenia in patients led we, and other, to hypothesize that the hematopoietic development might be abnormal in the FA embryo. Indeed, unlike adult hematopoietic stem cells (HSCs) which are quiescent in the BM niche, during embryonic life HSCs are in active proliferation in sites of expansion such as fetal liver and placenta, where they get amplified and acquire properties of adult HSC .We hypothesized that in FA, the FA defect in response to the replicative stress could impair the expension of the HSC pool.In order to investigate this hypothesis, we carried out studies in Fancg-/- knock out mice and in human FA fetuses obtained with informed consent from medical abortion. In Fancg-/- mice, FACS analysis revealed a 1,5- to 3-fold deficiency in hematopoietic stem and progenitor cells (HSPC) very early during embryonic development (i.e 11.5 days of gestation - E11.5) in fetal liver (FL) and placenta (Pl) (p <0.001). In both organs, this defect persists during the whole period of amplification (until E14.5 for FL and E12.5 for Pl). In vitro clonogenic assays also demonstrated a 2- fold defect in granulocyte, erythrocyte and macrophage (GEM) progenitors both in Fancg-/- FL or Pl compared to WT (p <0.001), and 4 to 5- fold defect in more immature mixed GEM progenitors in FL (p <0.001). LTC-IC frequency of the HSC-enriched Lineage- Sca1+ AA4.1+ population (LSA) of E14.5 Fancg-/- FL comforted this later result, since it was 5-fold lower than for WT. In vivo long-term hematopoietic reconstitution (LTR) assays confirmed a deficit of the HSC enriched LSA population of E14.5 Fancg-/- FL. Indeed, although the percentage of mice reconstituted was as good as that obtained with the same number of WT LSA, the CD45 Ly5.2 chimerism was reduced (49±20% vs 84±4% for 1000 LSA injected, and 56±12% vs 87±2% for 5000 LSA). Interestingly, bone marrow analysis of mice reconstituted with Fancg-/- LSA 22 weeks after injection showed a level of CD45 Ly5.2 chimerism 3-fold lower than that found in blood, spleen and thymus, as well as a very low chimerism for myeloid GEM lineages, contrasting with a high chimerism for B and T lymphoid lineages. Moreover, we were able to demonstrate that this deficit is already present at E12.5, both in Fancg-/- FL and Pl. Indeed, no mice reconstituted with 3.105 total Fancg-/- fetal liver cells, while 100% injected with the same number of WT FL cells got reconstituted with a chimerism of 59,5±5%. For Pl, when 500 000 cells were injected, reconstitution was observed in only 1 out of 3 mice for Fancg-/- (29% chimerism), and in 3 out of 3 mice for WT (88±4% chimerism). In human FA FL of 14 weeks of gestation, we also observed a 4-fold defect of HSPC with a total lack of in vitro amplification compared to control, in agreement with the mice data. Taken together, these data demonstrate that a profound deficit of HSCs and progenitors cells is present since the earlier stages of embryonic development in FA. In addition, using organotypic cultures of E11 aortas, we could show that this defect of amplification is already present in HSCs emerging from Fancg-/- aorta, which showed a 2-fold lower rate of amplification compared to WT. More importantly, our results show for the first time exhaustion in myeloid lineage of FA, in agreement with what is observed in children with FA disease. Altogether, our work suggests a role of the FA pathway during the development of the hematopoietic system leading to a deficit of amplification of HSC. Comparison of FA HSC transcriptome with that of control HSC in FL and Pl is in progress. It should allow to identify the key pathways involved in the embryonic HSC amplification that are deregulated in FA, and hopefully getting more insights in the pathogenesis of the BMF and leukemogenesis in FA patients. Disclosures No relevant conflicts of interest to declare.

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Impaired Bone Marrow Microenvironment in Fanconi Anemia Murine Model Is Responsible for the Defective Hematopoietic Stem/ Progenitor Cell Mobilization.

Blood ◽

10.1182/blood.v114.22.3629.3629 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3629-3629

Author(s):

Yan Li ◽

Shi Chen ◽

Yongzheng He ◽

Xiaohong Li ◽

Fengchun Yang

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Fanconi Anemia ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Bone Marrow Microenvironment ◽

Hematopoietic Stem

Abstract Abstract 3629 Poster Board III-565 Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by progressive bone marrow failure (BMF) and acquisition of malignancies. The only cure for BMF is a human leukocyte antigen (HLA)-matched BM transplantation from a family member or autologous stem cells before BMF develops. Therefore, mobilization of hematopoietic stem/progenitor cells (HSPCs) from BM into peripheral blood (PB) for collection has been a prerequisite for the therapy. However, patients with FA show a markedly decreased HSPC mobilization in response to the traditional mobilizing drug G-CSF and the mechanism(s) underlying the defect remains unknown. Mesenchymal stem/progenitor cells (MSPCs) have been known to be the common progenitor of a variety of cellular components in the bone marrow microenvironment. MSPCs express/secrete cytokines, extracellular matrix proteins and cell adhesion molecules, which regulate the homing, migration, proliferation and survival of HSPCs in vitro and in vivo. Recently, we reported that Fancg-/- MSPCs have a defect in hematopoietic supportive activity both in vitro and in vivo (Li et al. Blood, 2009). In the current studies, we show that Fancg-/- MSPCs have significant reduction in HSPC recruitment as compared to WT MSPCs in a transwell assay. Furthermore, Fancg-/- MSPCs have an alteration in the production of multiple cytokines/chemokines. Application of a neutralizing antibody to the cytokine blocked WT MSPC mediated HSPC migration in vitro. Furthermore, administration of the specific cytokine significantly increased HSPC mobilization in the Fancg-/- mice in vivo. These results demonstrated that an impaired BM microenvironment, specifically MSPCs in Fancg-/- mice, is contributory to defective HSPC mobilization. This study provides evidence of alternative clinical therapeutics for the mobilization of HSPCs in FA patients. Disclosures: No relevant conflicts of interest to declare.

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Deletion Of Fanca Or Fancd2 Dysregulates Treg Activity and Exacerbates Gvhd In Mice

Blood ◽

10.1182/blood.v122.21.3239.3239 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3239-3239

Author(s):

Wei Du ◽

Ozlem Erden ◽

Andrew Wilson ◽

Jared Sipple ◽

Jonathan Schick ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Transcriptional Activity ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

In Vitro Proliferation

Abstract Fanconi anemia (FA) is a genetic disorder associated with bone marrow failure and leukemia. Recent studies demonstrate fundamental immune defects in FA. However, the mechanisms that are critical for FA immunodeficiency are not known. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs) and exacerbates graft-versus-host disease (GVHD) in mice. Recipient mice of Fanca-/- or Fancd2-/- bone marrow chimeras exhibited severe acute GVHD after allogeneic bone marrow transplantation (BMT). Further study showed that T cells from Fanca-/- or Fancd2-/- mice induced higher GVHD lethality than those from WT littermates. Mechanistically, FA Tregs possessed lower proliferative suppression potential compared to WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25+Foxp3+ Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 transcriptional activity. Additionally, CD25+Foxp3+ Tregs of Fanca-/- or Fancd2-/- mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, incremental NF-kB transcriptional activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs desreased GVHD mortality. Our study uncovers an essential role for the FA proteins in maintaining Treg homeostasis and suggests that targeted blocking NF-kB signaling within T cells represents an attractive therapeutic strategy to ameliorate GVHD in FA. 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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Aberrant Type-2-Lactosaminoglycan Synthesis Severely Impairs Thrombopoiesis

Blood ◽

10.1182/blood.v122.21.2320.2320 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2320-2320

Author(s):

Silvia Giannini ◽

Antonija Jurak Begonja ◽

Max Adelmann ◽

Karin M. Hoffmeister

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fetal Liver ◽

White Blood Cells ◽

Bleeding Complications ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Platelet Production

Abstract Platelet recovery following myelosuppressive/myeloablative chemotherapy is crucial to avoid bleeding complications of cancer treatment. Platelets are produced by bone marrow megakaryocytes (MKs), which develop and mature from hematopoietic stem cells (HSC). Mature MKs interact with sinusoidal bone marrow endothelial cells to form transendothelial pseudopods called proplatelets from which platelets are released into the bloodstream. Platelet survival is dependent on correct glycan expression. We here investigate the role of Type-2-Lactosaminoglycans (Type-2-LacNAc) in platelet production. beta1,4Galactosyltransferase 1 (beta4GalT1) is a major enzyme involved in Type-2-LacNAc synthesis which adds Galactose (Gal) to terminal N-Acetylglucosamine (GlcNAc) to form beta1,4Gal-GlcNAc (Type-2-LacNAc).beta4GalT1 deficient mice die in uthero between E15.5 and E16.5. A small percentage of beta4GalT1-/- mice survive until adulthood and they have severe macrothrombocytopenia but normal platelet clearance. Lethally irradiated wild type mice transplanted with beta4GalT1 deficient fetal liver cells failed to produce circulating beta4GalT1 deficient platelets, in marked contrast to beta4GalT1 deficient white blood cells, despite beta4GalT1 deficient MKs have been detected in the bone marrow of transplanted mice. beta4GalT1 deficient fetal liver MKs poorly produce proplatelets in vitro, following their normal maturation and differentiation, as judged by number, morphology, ploidy and expression of main surface glycoproteins. Our data strongly support the notion that glycosylation mediated by beta4GalT1 is crucial for platelet production in vitro and in vivo and demonstrate for the first time a role for post-translational glycan modification in platelet production. Disclosures: No relevant conflicts of interest to declare.

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Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽

10.1182/blood.v95.2.700 ◽

2000 ◽

Vol 95 (2) ◽

pp. 700-704 ◽

Cited By ~ 40

Author(s):

Kimberly A. Gush ◽

Kai-Ling Fu ◽

Markus Grompe ◽

Christopher E. Walsh

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mitomycin C ◽

Fanconi Anemia ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Hematopoietic Stem ◽

Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

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Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

Stem Cells International ◽

10.1155/2020/8885154 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Huihong Zeng ◽

Jiaoqi Cheng ◽

Ying Fan ◽

Yingying Luan ◽

Juan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells

Blood ◽

10.1182/blood-2006-05-023028 ◽

2006 ◽

Vol 109 (5) ◽

pp. 1825-1833 ◽

Cited By ~ 46

Author(s):

Sinead Forde ◽

Britt Jorgensen Tye ◽

Sarah E. Newey ◽

Maria Roubelakis ◽

Jon Smythe ◽

...

Keyword(s):

Bone Marrow ◽

Mapk Signaling ◽

Hematopoietic Progenitor Cell ◽

Bone Marrow Niche ◽

Hematopoietic Stem ◽

Surface Receptors ◽

Cxcr4 Receptor ◽

Novel Association ◽

Pkc Ζ

Abstract Hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC) homing to specific microenvironmental niches involves interactions between multiple receptor ligand pairs. Although CXCL12/CXCR4 plays a central role in these events, CXCR4 regulators that provide the specificity for such cells to lodge and be retained in particular niches are poorly defined. Here, we provide evidence that the sialomucin endolyn (CD164), an adhesion receptor that regulates the adhesion of CD34+ cells to bone marrow stroma and the recruitment of CD34+CD38lo/− cells into cycle, associates with CXCR4. The class II 103B2 monoclonal antibody, which binds the CD164 N-linked glycan-dependent epitope or CD164 knockdown by RNA interference, significantly inhibits the migration of CD133+ HPCs toward CXCL12 in vitro. On presentation of CXCL12 on fibronectin, CD164 associates with CXCR4, an interaction that temporally follows the association of CXCR4 with the integrins VLA-4 and VLA-5. This coincides with PKC-ζ and Akt signaling through the CXCR4 receptor, which was disrupted on the loss of CD164 though MAPK signaling was unaffected. We therefore demonstrate a novel association among 3 distinct families of cell-surface receptors that regulate cell migratory responses and identify a new role for CD164. We propose that this lends specificity to the homing and lodgment of these cells within the bone marrow niche.

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The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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23 Years of Management: A Retrospective Review of Treatment for Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.1091.1091 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1091-1091

Author(s):

Connie M Piccone ◽

Marie Boorman Martin ◽

Zung Vu Tran ◽

Kim Smith-Whitley

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Retrospective Review ◽

Pediatric Patients ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Complete Response ◽

Primary Objective ◽

Hematopoietic Stem ◽

Transfusion Independence

Abstract Abstract 1091 Poster Board I-113 Introduction Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. In the past, AA was considered to be a fatal disease; however, current therapies, including bone marrow transplantation or immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CSA), are curative in the majority of patients. IST is effective at restoring hematopoietic stem cell production, but relapse and evolution to myelodysplastic syndromes remain clinical challenges. Additionally, there is no real consensus regarding optimal CSA levels, duration of CSA treatment, or the optimal use of growth factors and their relationship to the development of clonal disease. Objectives The primary objective was to review treatment management for severe AA in pediatric patients in order to elucidate treatment differences and review morbidity and mortality as they relate to treatment variation. Study Design/Methods A retrospective review of pediatric patients treated at the Children's Hospital of Philadelphia for AA (both severe and moderate) over a 23 year period was performed. Results A total of 70 patients with AA were treated at our institution from 1985 to July 2008. Exclusions included: 6 patients who received some type of initial treatment at outside institutions, 4 patients who had missing records, and 2 patients who had a diagnosis of moderate AA. Thus, a total of 58 patient records were included in the analysis. Of the total patients reviewed, 60% were male and 40% were female. 34.5% of patients were African-American, and 57% were diagnosed in 2000 or later. The mean age at diagnosis was 9.5±5.8 years. 52% fell into the category of very severe AA based on published diagnostic criteria, 45% had severe AA, and 2 patients (3%) had moderate AA. 15.5% of patients developed AA in the setting of acute hepatitis. More than half of the patients treated with IST had a complete response (CR). The average time to CR was 15±15 months. Average duration of CSA treatment was 15±13 months and 8.6±10.7 months for growth factor. Two patients (3.5%) died, one from complications unrelated to AA and one from infectious complications post-BMT after initial IST failure. Average time to transfusion independence for all patients was 8±11 months (with a range of 0-54 months). Not surprisingly, the time to transfusion independence was significantly associated with IST failure (p=0.010). Patients who failed treatment had an average time to transfusion independence of 17±16 months as compared to those who were complete responders who had an average time to transfusion independence of 3±3 months. Additionally, there was a significant association between IST failure and CSA levels (p=0.014). Patients who had nontherapeutic CSA levels overall had an increased rate of treatment failure. Of those patients who were nontherapeutic, 56% were noncompliant with CSA administration. There was no significant association between IST failure and bone marrow cellularity (p=0.251). PNH was diagnosed in 5% of patients; there were no patients with evidence of myelodysplastic syndrome (MDS). Two of the 3 patients with PNH failed initial IST. Another 2 patients had evidence of a cytogenetic abnormality (16q deletion), but never progressed to MDS. (Note: averages presented as mean±SD) Conclusions/Methods With current IST regimens, AA is curative in the majority of pediatric patients. IST failure was associated with nonadherence to CSA treatment. For patients with confirmed clonal disease, it is possible that IST failure and the ultimate development of clonal disease are related. Disclosures No relevant conflicts of interest to declare.

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