Mesenchymal Stromal Cell Irradiation Interferes with the Adipogenic/Osteogenic Differentiation Balance and Improves Their Hematopoietic-Supporting Ability

Abstract

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

“…In all the assays, MSCs were characterized according to the definition criteria established by ISCT and as previously described (Supporting Information Fig. S1).…”

“…BM mononuclear cells (BM‐MNC) were isolated by density‐gradient centrifugation (Ficoll‐Paque, density: 1.077 g/ml, GE Healthcare‐BioSciences). Cells were seeded at a density of 1 × 10 6 cells per centimeter square and expanded as previously described at 37°C and 5% of CO 2 in a humidified atmosphere in Dulbecco's modified Eagle's medium‐low glucose (Gibco, Life‐Technologies) with 10% fetal bovine serum (Gibco, Life Technologies) and 1% penicillin/streptomycin. After third passage, MSC were assessed according to the criteria proposed by the International Society for Cellular Therapy (ISCT) .…”

“…Data were analyzed using Infinicyt. Cells were considered to be in an early apoptotic state, late apoptosis, or dead if they were Annexin V + /7‐AAD − , Annexin V + /7‐AAD + , or Annexin V − /7‐AAD + , respectively, as previously reported (Supporting Information Fig. S3A).…”

Therapeutic Irradiation: Consequences for Bone and Bone Marrow Adipose Tissue

“…There is a decrease in trabecular bone volume, an increase in bone marrow adiposity (BMA), increased CTX/TRAP5 levels in the serum, and prolonged fracture healing times (2). Irradiation also depletes hematopoietic and skeletal stem cell populations within the BM (11–13). Bone marrow transplants allow for trabecular recovery, reduced BMA, and increased cell number within the BM microenvironment (12).…”

“…Bone marrow transplants allow for trabecular recovery, reduced BMA, and increased cell number within the BM microenvironment (12). Skeletal stem cells (SSCs), previously referred to as mesenchymal stem cells, appear to be effected by the irradiation source (photon irradiation vs. ionizing irradiation) and dose resulting in the varying differentiation potential observed in different in vitro studies (Figure 1) (11, 14–16). In murine models, the balance favors adipogenesis at the expense of osteogenesis, as a result of irradiation-induced bone loss.…”