Comparison of alternative mesenchymal stem cell sources for cell banking and musculoskeletal advanced therapies.

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs.

Animal models of multiple sclerosis.

Since its first description, experimental autoimmune encephalomyelitis, originally designated experimental allergic encephalitis (EAE), has been proposed as animal model to investigate pathogenetic hypotheses and test new treatments in the field of central nervous system inflammation and demyelination, which has become, in the last 30 years, the most popular animal model of multiple sclerosis (MS). This experimental disease can be obtained in all mammals tested so far, including nonhuman primates, allowing very advanced preclinical studies. Its appropriate use has led to the development of the most recent treatments approved for MS, also demonstrating its predictive value when properly handled. Some of the most exciting experiments validating the use of neural precursor cells (NPCs) as a potential therapeutic option in CNS inflammation have been performed in this model. We review here the most relevant immunological features of EAE in the different animal species and strains, and describe detailed protocols to obtain the three most common clinical courses of EAE in mice, with the hope to provide both cultural and practical basis for the use of this fascinating animal model.

Predicting poor peripheral blood stem cell collection in patients with multiple myeloma receiving pre-transplant induction therapy with novel agents and mobilized with cyclophosphamide plus granulocyte-colony stimulating factor: results from a Gruppo Italiano Malattie EMatologiche dell'Adulto Multiple Myeloma Working Party study.

INTRODUCTION: A still not well defined proportion of patients with multiple myeloma (MM) and eligible for autologous stem cell transplantation (AuSCT) fails to mobilize CD34+ peripheral blood stem cells (PBSC) at all or to collect an adequate number for a safe procedure or sufficient for multiple transplants. These so-called "poor-mobilizers" are difficult to be predicted, due to marked difference across previous heterogeneous studies. METHODS: We aimed to develop a method based on simple clinical parameters for predicting unsuccessful (<2×10(6)/kg) or sub-optimal (<5×10(6)/kg) collections of CD34+ PBSC in newly diagnosed MM patients eligible for AuSCT, treated with novel agents and receiving an homogeneous mobilizing therapy with cyclophosphamide and granulocyte-colony stimulating factor (G-CSF). To this purpose, 1,348 patients enrolled in five consecutive Italian clinical trials were retrospectively analysed. Age, baseline low peripheral blood cell counts, use of lenalidomide, and haematological toxicity developed during induction were taken into account as possible factors associated with poor mobilization. RESULTS: Overall, 280 patients (20.8%) showed either sub-optimal (167 patients, 12.4%) or unsuccessful (113 patients, 8.4%) collections. All analysed parameters negatively influenced the procedure, but only age and haematological toxicity during induction maintained their significance at multivariate analysis. Based on ordinal logistic regression model, we constructed a risk heat-map where the four parameters were pooled and weighted according to their relevance as single or combined variables. This model was predictive for different probabilities of failure, suboptimal or optimal outcomes. CONCLUSIONS: We found that about one fifth of newly diagnosed MM fails to collect an adequate number of PBSC. Our model, based on a large group of patients treated frontline with novel agents and receiving the most popular mobilizing approach currently employed in Europe, is applicable in individual subjects and may contribute to the early identification of "poor mobilizer" phenotypes.

Validation of vacuum-based refrigerated system for biobanking tissue preservation: analysis of cellular morphology, protein stability, and RNA quality.

Biobanks of fresh, unfixed human normal and malignant tissues represent a valuable source for gene expression analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis in both clinical and translational research is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. The aim of this study was to evaluate an innovative vacuum-based refrigerated system, as a logistically feasible technology to increase the collection of tissue specimens, preserving the integrity of cellular and molecular components. We tested randomly-selected tissues stored under vacuum at 4°C by using endpoints important for research and diagnosis, including tissue morphology, epitope stability, and RNA integrity. Gene expression was evaluated by qualitative and quantitative RT analysis of selected housekeeping and tissue-specific genes. Tissue morphology and overall protein stability were generally well preserved, being compromised only in gallbladder tissue. By contrast, phosphoprotein and RNA analysis demonstrated a time-dependent degree of degradation, with progressive loss of stability from 24 to 72 hours. However, this reduction in RNA quality did not represent a limitation for successful expression analysis of selected genes. Indeed, a comparative qualitative and quantitative RT-PCR analysis showed that RNA extracted from tissues stored under vacuum is suitable for gene expression profiling, but requires highly sensitive technologies, such as quantitative RT-PCR. These data suggest that the refrigerated vacuum-based system represents a suitable and feasible technology for routine transport of fresh specimens from surgery to biobanks, thus increasing the opportunity to collect biospecimens.