Following Autologous Stem Cell Transplantation (1 Yr) Mesenchymal Stem Cells Are Functionally Impaired with Reduced Hematopoietic Support

Autologous stem cell transplantation (ASCT) is frequently applied in patients with multiple myeloma and malignant lymphoma. Although adequate steady state hematopoiesis with normal peripheral blood counts is attained after ASCT, marked cytopenias may occur in times of stress such as sepsis or re-exposure to chemotherapy. Our group has previously shown impairment of the hematopoietic stem cell (HSC) compartment 1 year post ASCT (pASCT), reflected by reduced HSC frequency and quiescence, and increased ROS production (Haematologica 2013;98:1264). Considering the essential role for mesenchymal stem cells (MSCs) in supporting hematopoiesis, we studied the MSC compartment 1 year post ASCT.

Bone marrow biopsies from pASCT patients (n=17) were studied and compared to normal bone marrow from healthy donors (NBM, n=20) by performing immunohistochemistry staining of endothelial cells by CD34 (indicating microvessel density, MVD) and MSCs by nestin, CD146 (Melanoma Cell Adhesion Molecule, MCAM) and CD271 (Nerve Growth Factor Receptor, NGFR). A significant increase in CD271⁺ MSCs was observed in pASCT bone marrow biopsies compared to NBM (p<0.0001), while the expression of additional markers did not differ between pASCT vs. NBM. MSCs were cultured from the CD34^- fraction of bone marrow mononuclear cells, obtained from pASCT patients (n=17) and MSCs derived from NBM (n=20). MSCs were selected by their plastic-adherency and replated to generate MSCs. Although pASCT MSCs and NBM MSCs had similar population doubling times (1.92±0.22 and 3.52±1.02 in passage 4 (P4), pASCT MSCs cultured in vitro demonstrated a change in morphology from the onset of P4. We also observed premature exhaustion of growth in 45% of the studied patients at P5 (vs. 18% in NBM) and increased senescence shown by B-galactosidase staining in P5/P6 (p=0.04). Differentiation assays did not show impairment in differentiation towards osteoblasts or adipocytes of pASCT MSCs. Gene expression analysis on early passage MSCs showed upregulation of pro-inflammatory and cell cycle genes, such as IL6 and p21, in pASCT MSCs compared to NBM MSCs. Co-culture studies with cord blood-derived CD34⁺ cells on pASCT MSCs showed a significant reduction in output in CFC assays and significant reduction in number of cobblestone-area forming cells in pASCT co-cultures versus NBM (p < 0.05).

Given the higher incidence of MDS and AML after ASCT, we questioned whether the observed phenotype of pASCT MSCs resembles MSCs from patients with MDS and AML. Therefore the endothelial and mesenchymal compartments of MDS (n=20) and AML (n=23) patients were studied. An increase in MVD was detected in MDS/AML bone marrow biopsies in contrast to NBM and pASCT (p < 0.05), while the expression of CD146, CD271 and nestin in MDS/AML patients was not significantly increased. 25% of AML MSC cultures showed no growth in the first passage. When MSC growth did occur, the remaining cultures did not show a difference in population doubling time or expansion. However, a change in morphology of MDS/AML MSCs similar to pASCT MSCs was observed. Studies of early passages of MDS/AML MSCs demonstrated a significantly increased gene expression of IL-6 and p21 comparable to pASCT MSCs. In addition PITX2 and Foxc1 expression was increased but no difference was observed in pASCT vs. MDS/AML MSCs. PITX2 has been linked to increased senescence of MDS MSCs while Foxc1 is linked to adipo-osteoprogenitor cell differentiation thereby affecting the HSC compartment.

Since none of the pASCT patients did develop MDS, immunohistochemical stainings were also performed on bone marrow biopsies of patients that developed therapy related (t-)MDS/AML following ASCT for lymphoma and myeloma (n=7), after a mean of 117 (MDS) and 50 months (AML). An increase in MVD was observed shortly before or during MDS/AML development, which is probably related to the emergence of malignant cells. No major changes in the phenotype of the MSC compartment were observed before or during the emergence of t-MDS/AML, indicating that t-MDS/AML is preceded by an increase in MVD without distinct changes in the MSC compartment.

In summary our results demonstrate that MSCs are affected after ASCT, as shown by expression pattern and functionality. These changes result in a pro-inflammatory phenotype with premature senescence and impaired support of hematopoietic cells, which may account for the reduced bone marrow reserve observed in pASCT patients.