MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR MATRIX DISPLAYS
ALTERED STRUCTURE AND IMPAIRED FUNCTIONALITY IN MDS
Manja Wobus1, Anna Mies1, Valentina Magno2, Susann Winter3, Cindy Welzel1, Amanpreet Bains4,
Michael Cross5, Carsten Werner6, Martin Bornhäuser7 and Uwe Platzbecker8
(1)Department of Medicine 1, University Hospital TU Dresden, Dresden, Germany, (2)Max Bergmann
Center of Biomaterials, TU Dresden, Dresden, Germany, (3)Department of Medicine I, University
Hospital Carl Gustav Carus, Technische Universität, Dresden, Germany, (4)Medical Department I,
Haematology and Cell Therapy, University of Leipzig Medical Center, Leipzig, Germany, (5)Department
of Hematology and Oncology, Leipzig, Germany, (6)Max Bergmann Center of Biomaterials Dresden,
Dresden, Germany, (7)Medical Department 1, University Hospital Dresden, Dresden, Germany,
(8)Leipzig University Hospital, Leipzig, Germany
Objectives : The bone marrow microenvironment is involved in the deregulation of
hematopoiesis in myelodysplastic syndromes (MDS). Particularly, the extracellular matrix
(ECM) seems to be an interesting player since it regulates stem cell fate through the
modulation of growth factor binding, function and release. Here, we initiated a
comprehensive analysis of mesenchymal stromal cell (MSC) derived ECM from MDS patients
in comparison to healthy controls.
Methods : MSCs of MDS patients (n=8, IPSS low and intermediate risk) and age-adjusted
healthy donors were seeded on poly-octadecene-alt-maleic anhydride coated glass slides. To
yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning
electron microscopy (SEM), sulfated glycosaminoglycans (GAGs) staining (fluorescent
LectinWGA and LectinPNA) and quantification (Blyscan assay). Moreover, purified CD34+
HSPCs were cultured on healthy and MDS MSC derived ECM for 4 and 8 days, respectively.
Subsequently, we analyzed the HSPC expansion and their distribution as well as the
phenotype by flow cytometry and cytoskeletal arrangement by F-actin staining.
Results : ECM from MDS MSCs displayed a denser fiber meshwork with thicker bundles. We
could detect more abundant GAGs on MDS ECM, present as thin white flakes on ECM fibers
or as bigger white sponge-like structures. Fluorescent lectin staining and confocal imaging
demonstrated a higher ECM production in MDS patients. Quantification by Blyscan assay
confirmed these observations with significantly higher sulfated GAG concentrations in MDS
ECM. Moreover, staining intensities for fibronectin and collagen IV were higher in MDS ECM.
After seeding 5x104 purified CD34+ HSPCs, we detected significantly higher expansion factors
on ECM from healthy MSCs compared to MDS ECM and to plastic culture after 4 days
(19.2x104/ 9.9x104/ 6.2x104) and 8 days (18.5x104/ 13.4x104/ 14.1x104), respectively. Of
note, the proportion of adherent cells was higher on healthy than on MDS ECM (67.2% vs.
55.7%). Since cell-matrix interaction is mediated by integrins, we analyzed the surface
expression of CD41, CD51 and CD61. Within the adherent fraction on healthy ECM up to 50%
more cells were positive for all three integrin subunits when compared to supernatant cells.
Highest expression was shown for CD51 (ITGAV) which binds tenascin and fibronectin, both
important matrix proteins, thereby facilitating HSPC maintenance. F-actin staining
demonstrated a polarized morphology in HSPCs cultured on healthy ECM whereas cells on
MDS matrix displayed a round, non-polarized shape.
Conclusion : We provide first evidence for structural and functional alterations of MDS MSC
derived ECM which negatively impacts the support for hematopoiesis.
POSTER 10
SCIENTIFIC PROGRAMME
SESSION I
ETIOLOGY OF MDS
SESSION II
BIOLOGY OF MDS –
GENETIC ABNORMALITIES
SESSION III
BIOLOGY OF MDS:
STEM CELLS AND THE
MICROENVIRONMENT
SESSION IV
DIAGNOSTIC WORKUP
AND PROGNOSTIC
FACTORS IN MDS
SESSION V
SPECIFIC SUBTYPES
OF MDS, BASED ON
MORPHOLOGY AND
MOLECULAR BIOLOGY
SESSION VI
TREATMENT OF MDS
SESSION VII
CURRENT PROGRESS IN
THE TREATMENT OF MDS
SESSION VIII
FUTURE TREATMENTS
AND TREATMENT
STRATEGIES IN MDS
SESSION IX
LATE-BREAKING TALKS
SELECTED ABSTRACTS
FOR AN ORAL
PRESENTATION
SELECTED ABSTRACTS
AS E-POSTERS
DISCLOSURES