Brigitte Rüster

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow‐derived MSCs are able to differentiate, in vitro, into functional epithelial‐like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin‐like growth Factor‐II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT‐PCR (Taq‐man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial‐like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.

The AF4.MLL fusion protein is capable of inducing ALL in mice without requirement of MLL.AF4.

The chromosomal translocation t(4;11)(q21;q23) is the most frequent genetic aberration of the human MLL gene, resulting in high-risk acute lymphoblastic leukemia (ALL). To elucidate the leukemogenic potential of the fusion proteins MLL.AF4 and AF4.MLL, Lin(-)/Sca1(+) purified cells (LSPCs) were retrovirally transduced with either both fusion genes or with MLL.AF4 or AF4.MLL alone. Recipients of AF4.MLL- or double-transduced LSPCs developed pro-B ALL, B/T biphenotypic acute leukemia, or mixed lineage leukemia. Transplantation of MLL.AF4- or mock-transduced LSPCs did not result in disease development during an observation period of 13 months. These findings indicate that the expression of the AF4.MLL fusion protein is capable of inducing acute lymphoblastic leukemia even in the absence of the MLL.AF4 fusion protein. In view of recent findings, these results may imply that t(4;11) leukemia is based on 2 oncoproteins, providing an explanation for the very early onset of disease in humans.

NAT screening of blood donors for severe acute respiratory syndrome coronavirus can potentially prevent transfusion associated transmissions.

BACKGROUND: The severe acute respiratory syndrome (SARS) was first described in February 2003. Close contact with symptomatic patients appears to be the main route of transmission, whereas blood transfusion trans‐ mission could not be ruled out.

Erythrocytic precursor cells show potent shear stress resistant adhesion and home to hematopoietic tissue in vivo

BACKGROUND: Transfusion of erythropoietic precursor cells has been suggested as an alternative to conventional red blood cells. However, little is known about the fate of transfused erythrocytic precursors after they enter the bloodstream.

Beta-Chemokine CCL15 Affects the Adhesion and Migration of Hematopoietic Progenitor Cells.

Background: Hematopoietic stem and progenitor cell (HPC) motility is essential for HPC transplantation. The chemokine CXCL12 is key for HPC motility. Further regulators are of interest to improve HPC transplantation and regenerative medicine. Here the impact of the human chemokine CCL15 on HPC motility was investigated. Methods: CCL15 plasma concentrations were determined during HPC mobilization in humans. Activity of CCL15 on HPCs was investigated in murine assays, including chemotaxis, adhesion, and CFU-A assays, and competitive repopulation assays. Results: During HPC mobilization with granulocyte colony-stimulating factor, blood plasma contains increased concentrations (1.1 ± 0.1 ng/ml) of activated CCL15(27-92) versus 0.4 ± 0.1 ng/ml in controls (p = 0.02). CCL15(27-92) significantly enhanced CXCL12-induced transwell migration of Lin-/Sca1+ HPCs and strengthened shear stress-dependent adhesion to vascular cell adhesion molecule-1 (VCAM-1). CCL15(27-92) dose-dependently reduced the colony size in CFU-A assays performed with murine bone marrow and Lin-/Sca1+ HPCs. CCL15(27-92) did not show a direct impact on cell cycle status of HPCs. In murine repopulation assays, pretreatment of bone marrow with CCL15(27-92) significantly increased competitive repopulation. Conclusion: Our results point to a regulation of HPCs by CCL15 by modulating migratory and adhesive properties of HPCs with the potency to improve HPC short-term engraftment in stem cell transplantation.

Novel Role of Ras-GTPase Activating Protein SH3 Domain-Binding Protein G3BP in Adhesion and Migration of 32D Myeloid Progenitor Cells

Rho GTPases are involved in homing and mobilization of hematopoietic stem and progenitor cells due to their impact on cytoskeleton remodeling. We have previously shown that inhibition of Rho, Rac and Cdc42 clearly impairs ad- hesion of normal and leukemic hematopoietic progenitor cells (HPC) to fibronectin and migration in a three-dimensional stromal cell model. Here, we identified the Ras GTPase-Activating Protein SH3 Domain-Binding Protein (G3BP) as a target gene of Rho GTPases and analysed its role in regulating HPC motility. Overexpression of G3BP significantly enhanced adhesion of murine 32D HPC to fibronectin and human umbilical vein endothelial cells, increased the proportion of adherent cells in a flow chamber assay and promoted cell migration in a transwell assay and a three-dimensional stromal cell model suggesting a strong impact on the cytoskeleton. Immunofluorescent staining of G3BP-overexpressing fibroblasts revealed a Rho-like phenotype characterized by formation of actin stress fibers in contrast to the Rac-like phenotype of control fibroblasts. This is the first report implicating a role for G3BP in Rho GTPase-mediated signalling towards adhesion and migration of HPC. Our results may be of clinical importance, since G3BP was found overexpressed in human cancers.

Mice Deficient in Rap1A GTPase Display Reduced Adhesion of Hematopoietic and Mesenchymal Stromal Cells but Intact Hematopoietic Repopulation Potential

Background: The Guanine Triphosphatase (GTPase) Rap1A has been implicated in cell proliferation and migration in different tissues. Its role in the production, adhesion and migration of bone marrow-derived hematopoietic cells is however incompletely understood. Methods: We analysed mice lacking one or both Rap1A alleles and measured their adhesion function and their hematopoietic repopulation capacity using competitive bone marrow transplantation in the CD45.1/CD45.2 transplant model. We further employed flow cytometry and shear stress-dependent flow chamber adhesion assays for phenotypic characterization and analysis of adhesion functions. Results: Rap1A-/- mice showed decreased numbers of lymphocytes and hematopoietic colony forming progenitors in the blood, while the numbers of granulocytes, monocytes or platelets were not significantly altered compared to controls. Chemokine CXCL12-induced adhesion of lineage marker depleted (Lin-) bone marrow cells to Vascular Cell Adhesion Molecule (VCAM)-1 and to Intercellular Adhesion Molecule (ICAM)-1 was significantly reduced in Rap1A-/- mice. However, competitive repopulation by Rap1A deficient bone marrow cells was not inferior to wild type bone marrow 9 months after transplantation. No outgrowth of Mesenchymal Stromal Cells (MSCs) was observed from bone marrow of Rap1A-/- mice, whereas Rap1A+/- MSCs displayed reduced expression of adhesion molecules involved in progenitor homing and impaired adhesion to primary endothelial cells and VCAM-1 under shear stress in vitro, as compared to their wild-type counterparts. Conclusions: Rap1A regulates adhesion of immature hematopoietic cells and mesenchymal stromal cells, but appears to be redundant for hematopoietic engraftment.