Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. METHODS: MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. RESULTS: The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. CONCLUSIONS: The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells.

Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17ß-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.