Regulation of megakaryocytopoiesis in an in vitro stroma model: Preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

Objective. Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. Materials and Methods. Human CD34+ cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. Results. After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the β1 integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. Conclusion. In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite. Copyright (C) 2000 International Society for Experimental Hematology

Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells

Stromal cell-derived factor-1 (SDF-1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF-1-mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG-1 (KG-1v) was used, displaying CXCR4-dependent transmigration. Cell surface PGs on bone marrow endothelial cell line 4LHBMEC did not mediate SDF-1-induced transendothelial migration. In contrast, transwell filters precoated with various glycosaminoglycans (GAGs) enhanced migration toward SDF-1. SDF-1-induced migration was reduced by degradation of heparan sulfate in subendothelial matrix produced by 4LHBMEC. The stimulating effect of GAGs was caused by the formation of a stable haptotactic SDF-1 gradient, as SDF-1 bound to immobilized GAGs and triggered migration. Soluble heparan sulfate enhanced SDF-1-induced migration dose-dependently, suggesting that SDF-1-heparan sulfate complexes optimized SDF-1 presentation. In conclusion, we provide evidence that PGs in the subendothelial matrix establish an SDF-1 gradient guiding migrating HPC into the bone marrow

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions

Granulocyte colony-stimulating factor mobilized whole blood containing over 0.3 × 106/kg CD34+ cells is a sufficient graft in autologous transplantation for relapsed non-Hodgkin's lymphoma

The feasibility of unprocessed, granulocyte colony-stimulating factor (G-CSF)-mobilized whole blood (WB) as an alternative stem cell source for autologous stem cell transplantation was studied. Forty-seven relapsed non-Hodgkin's lymphoma (NHL) patients entered the study. After two or three ifosfamide, methotrexate and etoposide (IMVP) courses, 1 l of G-CSF-mobilized WB was collected and stored refrigerated for 72 h. Meanwhile, BAM conditioning was given: BCNU (carmustine) 300 mg/m(2), high-dose cytarabine 6000 mg/m(2) and melphalan 140 mg/m(2). Toxicity, haematological recovery and survival were assessed and compared with peripheral blood stem cell transplantation (PBSCT) and bone marrow transplantation (BMT) reference groups. High-dose G-CSF (2 x 12 microg/kg/d) gave the best mobilization results. Haematological recovery was related to the WB CD34+ content. A CD34+ threshold of >or= 0.3 10(6)/kg, obtained in 90% of patients using high-dose G-CSF, correlated with adequate recovery: absolute neutrophil count (ANC) > 0.5 x 10(9)/l: median 12 d (range 9-19). Platelet recovery > 20 and > 50 x 10(9)/l was 19 (11-59) and 30 d (14 not reached) respectively. Overall survival of patients or= 0.3 x 10(6)/kg CD34+ cells after BAM conditioning is a safe procedure, and offers a fully equivalent and less costly alternative for PBSC

Homing and clonogenic outgrowth of CD34+ peripheral blood stem cells: A role for L-selectin?

Objective. After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin+ stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. Materials and Methods. Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34+ cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin+L-selectin- cells or in the presence of antibodies. Results. Blocking of L-selectin on CD34+ cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34+ cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. Conclusion. Using in vitro assays for CD34+ stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc

Subendothelial Heparan Sulfate Proteoglycans Become Major L-Selectin and Monocyte Chemoattractant Protein-1 Ligands upon Renal Ischemia/Reperfusion

Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal ischemia/reperfusion, associated with severe endothelial damage, microvascular basement membrane (BM) heparan sulfate proteoglycans (HSPGs) are modified to bind L-selectin and monocyte chemoattractant protein-1. In an in vitro rolling and adhesion assay, L-selectin-binding HSPGs in artificial BM induced monocytic cell adhesion under reduced flow. We examined the in vivo relevance of BM HSPGs in renal ischemia/reperfusion using mice mutated for BM HSPGs perlecan (Hspg2Δ3/Δ3), collagen type XVIII (Col18a1−/−), or both (cross-bred Hspg2Δ3/Δ3×Col18a1−/−) and found that early monocyte/macrophage influx was impaired in Hspg2Δ3/Δ3×Col18a1−/− mice. Finally, we confirmed our observations in human renal allograft biopsies, showing that loss of endothelial expression of the extracellular endosulfatase HSulf-1 may be a likely mechanism underlying the induction of L-selectin- and monocyte chemoattractant protein-1-binding HSPGs associated with peritubular capillaries in human renal allograft rejection. Our results provide evidence for the concept that not only endothelial but also (microvascular) BM HSPGs can influence inflammatory responses

Identification of distinct prognostic subgroups in low- and intermediate-1-risk myelodysplastic syndromes by flow cytometry

The World Health Organization (WHO) classification contributes to refined classification and prognostication of myelodysplastic syndromes (MDSs). Flow cytometry might add significantly to diagnostic and prognostic criteria. Our analysis of bone marrow samples from 50 patients with MDS showed aberrant expression of differentiation antigens in the myelomonocytic lineage. This also accounted for refractory anemia (RA) with or without ringed sideroblasts (RS), indicating multilineage dysplasia. In 38% of patients, CD34+myeloid blasts expressed CD5, CD7, or CD56. Flow cytometry data were translated into a numerical MDS flow-score. Flow-scores increased significantly from RA with or without RS, refractory cytopenia with multilineage dysplasia (RCMD) with or without RS up to refractory anemia with excess of blasts-1 (RAEB-1) and RAEB-2. No significant differences were observed between WHO cytogenetic subgroups. Flow-scores were highly heterogeneous within International Prognostic Scoring System (IPSS) subgroups. Patients in progression to advanced MDS or acute myeloid leukemia had a significantly higher flow-score compared with non-transfusion-dependent patients. In 60% of patients with transfusion dependency or progressive disease, myeloid blasts expressed CD7 or CD56, in contrast to only 9% of non-transfusion-dependent patients. Moreover, all patients with pure RA with or without RS with aberrant myeloid blasts showed an adverse clinical course. In conclusion, flow cytometry in MDS identified aberrancies in the myelomonocytic lineage not otherwise determined by cytomorphology. In addition, flow cytometry identified patients at risk for transfusion dependency and/or progressive disease independent of known risk groups, which might have impact on treatment decisions and the prognostic scoring system in the near future