Abstract
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.
Highlights
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients
After exclusion of hematopoietic cells ( CD45+), endothelial precursor cells (CD31 +), and erythrocyte precursor cells (CD235a+), cells were gated on the CD271+ population, which is currently thought to comprise most of the primary BM stromal cells (BMSC) p opulation[13]
Significant knowledge has been generated through functional analysis of cultured BMSC isolated from whole bone marrow (BM) samples using their ability to adhere to plastic surfaces like culture d ishes[14]
Summary
Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. To study primary BMSC and interrogate their adipo-osteogenic differentiation potential, we developed a stringent flow cytometric sorting protocol for prospective isolation of rare CFU-F some of which give rise to multipotent BMSC from cryopreserved whole BM samples of healthy BM donors and newly diagnosed or currently untreated MDS and AML patients. This sorting procedure yields a population of cells with a homogeneous phenotype, the functional potential of these cells may still vary. We performed further functional analyses of this enriched population, through culture-expansion of CFU-F-derived BMSC and subsequent examination of their growth, differentiation, clonogenic capacity, and gene expression from BM samples of MDS and AML patients in comparison to age-matched healthy controls
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