Multi-potent progenitors in freshly isolated and cultured human mesenchymal stem cells: a comparison between adipose and dermal tissue.

Mesenchymal stem cells (MSCs) from human adult adipose tissue (A-MSCs) have a better differentiative ability than MSCs derived from the derma (D-MSCs). To test whether this difference is associated with differences in the content of multi-potent progenitors in A-MSCs, the number and the differentiative properties of multi-potent progenitors have been analyzed in various preparations of A-MSCs and D-MSCs. Adipogenic and osteogenic differentiation performed on colony-forming units have revealed that adipogenic and osteogenic progenitors are similar in the two populations, with only a slighty better performance of A-MSCs over D-MSCs from passages p0 to p15. An analysis of the presence of tri-, bi-, uni- and nulli-potent progenitors isolated immediately after isolation from tissues (p0) has shown comparable numbers of tri-potent and bi-potent progenitors in MSCs from the two tissues, whereas a higher content in uni-potent cells committed to adipocytes and a lower content in nulli-potent cells has been observed in A-MSCs. Furthermore, we have characterized the progenitors present in A-MSCs after six passages in vitro to verify the way in which in vitro culture can affect content in progenitor cells. We have observed that the percentage of tri-potent cells in A-MSCs at p6 remains similar to that observed at p0, although bi-potent and uni-potent progenitors committed to osteogenic differentiation increase at p6, whereas nulli-potent cells decrease at p6. These data indicate that the greater differentiative ability of A-MSC populations does not correlate directly with the number of multi-potent progenitors, suggesting that other factors influence the differentiation of bulk populations of A-MSCs.

Pluripotency regulators in human mesenchymal stem cells: expression of NANOG but not of OCT-4 and SOX-2.

Mesenchymal stem cells (MSCs) are adult multipotent cells able to differentiate toward mature mesodermal lineages. In spite of more than a decade of investigation, little is known about the molecular mechanisms regulating the undifferentiated state and the identity of distinct functional subpopulations in these cells. Transcription factors that regulate the maintenance of the pluripotent state in embryonic stem cells, including NANOG, SOX2, and OCT4, have been proposed to play a similar role also in adult stem cells, although with conflicting results. We performed a critical evaluation of expression of these 3 transcription factors and found that NANOG, but not OCT-4 and SOX-2, is expressed in cultured human adult MSCs. Actually, NANOG was not expressed in freshly isolated MSCs, but was detected only after in vitro culture. NANOG was detected only in proliferating cells, but not in MSCs induced to differentiate. The percentage of cells expressing NANOG was maintained throughout early passages of MSCs, but then started to decrease in late passages in MSCs from adipose tissue and heart but not from bone marrow. However, the number of NANOG-expressing cells did not associate with the proliferative and differentiative capabilities of MSC populations, neither its expression appeared to identify cells having stem or progenitor cell properties. Accordingly, we propose that activation of NANOG expression in MSCs is associated with, although cannot directly regulate, the transition from in vivo quiescence to adaptation to in vitro growth conditions.

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum.

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11beta-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP(+)-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11beta-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11beta-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

Intratumoral NAMI-A treatment triggers metastasis reduction, which correlates to CD44 regulation and tumor infiltrating lymphocyte recruitment.

Intratumor (i.t.) injection of 35 mg/kg/day NAMI-A for six consecutive days to CBA mice bearing i.m. implants of MCa mammary carcinoma reduces primary tumor growth and particularly lung metastasis formation, causing 60% of animals to be free of macroscopically detectable metastases. The i.t. treatment allows study of the effects of NAMI-A on in vivo tumor cells exposed to millimolar concentrations for a relatively prolonged time. Under these conditions, NAMI-A reduces the number of CD44+ tumor cells and changes tumor cell phenotype to a lower aggressive behavior, as shown by scanning electron microscopy analysis. On primary tumor site, NAMI-A causes unbalance between 2n and aneuploid cells in favor of lymphocytes. Furthermore, in tumor tissue, nitric oxide production is increased and active matrix metalloproteinase 9 is decreased, and these effects are accompanied by a reduced hemoglobin concentration. These data are in agreement with the reduction of tumor invasion and metastasis and suggest the therapeutic usefulness of NAMI-A in neoadjuvant or tumor reduction treatments for preventing metastasis formation. These data further stress the usefulness of intratumor treatments as experimental preclinical model for studying in vivo the mechanism of tumor cell interactions after prolonged exposure to ruthenium-based compounds to be developed for metastasis inhibition.

Distinct effects of dinuclear ruthenium(III) complexes on cell proliferation and on cell cycle regulation in human and murine tumor cell lines.

We have examined the biological and antitumor activity of a series of dinuclear ruthenium complexes. The aim of this study was to compare the in vitro effects of these new compounds on cell proliferation, cell distribution among cell cycle phases, and the expression of some proteins involved in cell cycle regulation. Results obtained show a mild cytotoxic activity against human and murine cell lines, more evident after prolonged exposure of cell challenge. Two of the eight dinuclear complexes [namely, compounds D3 (Na(2)[(RuCl(4)(dmso-S))(2)(mu-bipy)]) and D7 ([NH(4)][(RuCl(4)(dmso-S))(mu-pyz)(RuCl(3)(dmso-S)(dmso-O))]) modify cell cycle distribution similarly to imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate (NAMI-A), whereas the others have a low or negligible effect on this parameter. If we correlate the induction of cell cycle modifications with ruthenium uptake by tumor cells and with the modulation of proteins regulating cell cycle, we may stress that the induction of G(2)-M cell cycle arrest is related to the achievement of a threshold concentration of ruthenium inside the cells, which is dependent on the cell line being used, and that only cyclin B, among cell cycle regulating proteins examined by immunoblotting assays, appears to be significantly modified. This in vitro study shows that dinuclear ruthenium complexes may have a behavior similar to that of the monomer NAMI-A. These results encourage the future experimentation of their pharmacological properties in in vivo models.

Ruthenium-based NAMI-A type complexes with in vivo selective metastasis reduction and in vitro invasion inhibition unrelated to cell cytotoxicity.

A series of analogues of NAMI-A, a reference compound active on solid tumor metastases, were synthesized (NAMI-A type complexes). They share the same chemical structure of NAMI-A, and differ from it in the nature of the coordinated nitrogen ligand, such as pyrazole, thiazole and pyrazine, which are less basic than imidazole. This modification confers to the new NAMI-A type complexes a better stability in aqueous solution compared to the parent compound, a very important characteristic for a class of compounds that, with NAMI-A, is currently completing a phase I clinical trial at the Netherlands Cancer Institute of Amsterdam. Cytotoxicity and the effects on cell cycle and invasion were investigated on TS/A, B16-F10 and MCF-7 tumor cell lines, while the inhibition of lung metastases was determined on the mouse experimental tumors Lewis lung carcinoma and MCa mammary carcinoma. The new complexes show a pharmacological activity very similar to that of the parental compound NAMI-A: in vitro they are devoid of meaningful cytotoxicity against tumor cells, and in vivo they inhibit metastasis formation and growth approximately to the same extent as NAMI-A. Thus the new NAMI-A type complexes retain the same potent characteristic of NAMI-A to selectively interact with solid tumor metastases. However, compared to NAMI-A they do not stop cell cycle progression at G2-M level and are more active in preventing the spontaneous invasion of Matrigel by tumor cells exposed for 1 h to 10(-4) M concentration. Globally, these complexes take advantage of the knowledge on NAMI-A and appear particularly interesting for future clinical handling and applications.