Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy.

Ex vivo expansion and manipulation of human mesenchymal stem cells are important approaches to immunoregulatory and regenerative cell therapies. Although these cells show great potential for use, issues relating to their overall nature emerge as problems in the field. The need for extensive cell quantity amplification in vitro to obtain sufficient cell numbers for use, poses a risk of accumulating genetic and epigenetic abnormalities that could lead to sporadic malignant cell transformation. In this study, we have examined human mesenchymal stem cells derived from bone marrow, over extended culture time, using cytogenetic analyses, mixed lymphocyte reactions, proteomics and gene expression assays to determine whether the cultures would retain their potential for use in subsequent passages. Results indicate that in vitro cultures of these cells demonstrated chromosome variability after passage 4, but their immunomodulatory functions and differentiation capacity were maintained. At the molecular level, changes were observed from passage 5 on, indicating initiation of differentiation. Together, these results lead to the hypothesis that human mesenchymal stem cells cultures can be used successfully in cell therapy up to passage 4. However, use of cells from higher passages would have to be analysed case by case.

LLL-3, a STAT3 inhibitor, represses BCR-ABL-positive cell proliferation, activates apoptosis and improves the effects of Imatinib mesylate.

PURPOSE: The chimeric protein BCR-ABL, a constitutively active protein-tyrosine kinase, triggers downstream signalling proteins, such as STAT3, ultimately resulting in the survival of myeloid progenitors in BCR-ABL-positive leukemias. Here, we evaluated the effect of LLL-3, an inhibitor of STAT3 activity, on cell viability and its addictive effects with Imatinib mesylate (IM) treatment in BCR-ABL-positive cells. METHODS: Viability of cell lines was determined using the WST-1 assay in response to drug treatment, either LLL-3 alone or in conjunction with IM. Annexin V-FITC/PI staining, sub-G1 DNA content and Caspase-3/7 activation assays were performed to evaluate apoptosis. RESULTS: LLL-3 treatment decreased cell viability, triggered apoptosis and activated Caspases-3/7 in K562 cells. LLL-3 increases IM treatment to inhibited cell viability and activation of apoptosis in BCR-ABL-positive cell lines. CONCLUSIONS: LLL-3 reduced cell viability and induced apoptosis in K562 cells. Moreover, the observed addictive effects of co-treatment with IM and LLL-3 suggest this combination has therapeutic potential.