Are mesenchymal stromal cells from children resistant to apoptosis?

OBJECTIVES: Mesenchymal stromal cells (MSC) represent a novel cellular candidate in the field of transplantation and tissue regeneration. Their clinical application requires their in vitro expansion. The aim of this study was to assess the effect of conditions that would favour apoptosis, and of long-term expansion, on the characteristics of MSC from children. MATERIALS AND METHODS: Bone marrow mononuclear cells were cultured for 10 passages (P1-P10). Expression of CD105, CD146, CD95 and apoptosis by 7-amino-actinomycin D staining were evaluated. CFU-F and cell doubling time (DT) were assessed in every passage. Cell-cycle study was performed at P2 and P6. RESULTS: CFU-F decreased from 38 +/- 3.7 at P2 to 9.6 +/- 3.2 per 10 MSC/cm(2) at P10 and DT increased from 1.93 +/- 0.1 (P2) to 6.1 +/- 2.45 days (P10). A low percentage of apoptotic (dead) cells was detected at P2 and this did not change until P10. Cells at P2 were at G(0)/G(1) phase, but in advanced passages more cells were in an active state. Induction of apoptosis (addition of anti-Fas agonist antibody) using standard culture conditions, showed a minor effect on MSC survival. Serum deprivation of MSC (up to 72 h) revealed no substantial apoptotic effect while cells retained their tri-lineage differentiation capacity. CONCLUSIONS: We conclude that MSC from children retain their functional characteristics throughout serial passages and remain stable under conditions that usually cause apoptosis. These features render MSC, especially those of early passages, optimal candidates for use in clinical applications.

Adhesion molecules, endogenous granulocyte colony-stimulating factor levels and replating capacity of progenitors in autoimmune neutropenia of childhood.

AIM: To investigate the role of granulocyte colony-stimulating factor (G-CSF) and adhesion molecules and the response of bone marrow to peripheral cytopenia in autoimmune neutropenia of childhood (AIN). METHODS: Thirty-five children with AIN, 25 with acute leukaemia in remission, 10 of whom developed chemotherapy-associated neutropenia, and 28 non-neutropenic age-matched children were studied. The methods included haemopoietic progenitor cells' colony growth, replating of colony-forming unit-granulocyte macrophage (CFU-GM) of the 7th day and ELISA for the detection of serum levels of cytokines and adhesion molecules. RESULTS: In cases of severe autoimmune neutropenia, haemopoietic progenitors showed increased proliferative capacity compared to the control group (p = 0.03). Intercellular adhesion molecule-1 (ICAM-1), E-selectin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels inversely correlated with neutrophil counts (r = -0.8, p < 0.001 for ICAM-1, r = -0.5, p = 0.04 for E-selectin, r = -0.58, p = 0.01 for TNF-alpha, r = -0.62, p = 0.04 for IL-1beta). Serum ICAM-1, TNF-alpha and IL-1beta levels correlated positively with G-CSF levels (r = 0.47, p = 0.03 for ICAM-1, r = 0.65, p = 0.01 for TNF-alpha, r = 0.67, p = 0.04 for IL-1beta). Serum G-CSF levels were widely distributed and did not correlate with neutrophil counts (r = -0.44, p = 0.09). In secondary neutropenias the respective levels were lower than those in autoimmune neutropenia. CONCLUSIONS: Haemopoietic progenitors show increased proliferative capacity in cases of severe autoimmune neutropenia. G-CSF seems to act as an inducer of endothelium activation. The degree of neutropenia correlates with serum ICAM-1, E-selectin, TNF-alpha and IL-1beta levels, indicating the existence of an activated endothelium and presumably of a latent, low-grade, inflammatory process in severe autoimmune neutropenia.