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.

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Normal and leukemic blood cell progenitors depend upon the bone marrow (BM) stroma with which they communicate through soluble and membrane-anchored mediators, adhesive interactions and gap junctions (GJ). Regarding hematopoiesis, it is believed that it can be influenced by connexin expression, but the exact role of GJ in cell death and proliferation is not clear. Using flow cytometry, we monitored the division rate of leukemic cell lines, communicating and not communicating with stromal cell line through GJ. We found that GJ-coupled cells (i) did not proliferate; (ii) were kept in G0; and (iii) were protected from drug-induced apoptosis when compared to either total or uncoupled cell population. We conclude that GJ coupling between stroma and leukemic lymphoblasts prevents proliferation, keeping cells in a quiescent state, thus increasing their resistance to antimitotic drugs. Since GJ are particularly abundant in the sub-endosteal environment, which harbors blood stem cells, we also asked which cells within the normal human BM communicate with the stroma. Using a primary BM stroma cell culture, our results show that 80% of CD34 þ progenitors communicate through GJ. We propose that blood cell progenitors might be retained in the low-cycling state by GJ-mediated communication with the hematopoietic stroma.

In the general population, about 5% of individuals are homozygotic and 35% are heterozygotic carriers for chitotriosidase (ChT) deficiency. Activated macrophages are considered to be the main source of plasma ChT activity, which permits the biochemical characterization of homozygote deficients. However, in the case of detecting heterozygotic carriers, the results are often inconclusive. The activities of ChT in plasma and mononuclear (MN) and polymorphonuclear (PMN) leukocytes were determined in 169 control subjects (72 males and 97 females) with a mean age (+/- SD) of 47.5+/-9.7 years (range 18-96 years). The specific enzyme activity was in PMN leukocytes >MN leukocytes >plasma, with a highly significant partial correlation being found between the activities of ChT in plasma and PMN leukocytes (r=0.578, P<0.001). A significant correlation was found between the age of the patients studied and plasma ChT activity (r=0.568, P<0.001). No significant correlation was found for enzyme activities in MN (r=0.105) or in PMN leukocytes (r=0.043). The results obtained suggest that, in normal physiological conditions, PMN leukocytes may secrete ChT to the plasma. Although the activities of ChT in MN and PMN leukocytes are not affected by demographic factors, it is not possible to use them for the biochemical detection of ChT-deficient heterozygotic carriers.