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.

Study of oncogenic transformation in ex vivo expanded mesenchymal cells, from paediatric bone marrow.

OBJECTIVES: Mesenchymal stromal cells (MSCs) have attracted considerable interest in both the scientific and clinical fields. In order to obtain a sufficient cell number for application, their in vitro expansion is necessary, but during this process their characteristics may be altered and cells may acquire oncogenic properties. We have investigated properties of MSC that may be related to oncogenesis, a critical parameter that has to be evaluated prior to MSC clinical use. MATERIALS AND METHODS: We studied the expression of p53, p16, RB, H-RAS and human telomerase reverse transcriptase (hTERT) in MSCs from bone marrow of children diagnosed with idiopathic thrombocytopenic purpura (ITP) and autoimmune neutropenia. The same cells were seeded in soft agar to confirm their anchorage dependence and were karyotypically analysed. Finally, MSCs were subcutaneously transplanted into SCID mice and their ectopic osteogenic as well as tumorigenic potential was evaluated. RESULTS: We have shown that MSCs derived from bone marrow of children with ITP and autoimmune neutropenia do not undergo transformation, the cells expressed normal levels of p53, p16, RB and H-RAS. Expression of hTERT was undetectable, chromosome content remained stable, and their anchorage dependence was confirmed. In an in vivo model, when MSCs were subcutaneously transplanted into SCID mice, no tumorigenesis was observed. CONCLUSIONS: These findings suggest that MSCs from bone marrow of children do not have oncogenic properties and, therefore, represent validate candidates for applications in regenerative medicine.

Could cord blood be a source of mesenchymal stromal cells for clinical use?

BACKGROUND: Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. METHODS: Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated. RESULTS: Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. DISCUSSION: These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.

Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases.

BACKGROUND: Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. METHODS: BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. RESULTS: MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. DISCUSSION: MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.