Background Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. Methods and Findings UCB-derived mononuclear cells (MNC) or selected CD34+ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34+ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin+, CD133+ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. Conclusions This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.
Abstract Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.
Vitrinite reflectance and illite crystallinity of a great number of samples of fine-grained Mesozoic sediments from the Northern Calcareous Alpes between Allgäu and the Karwendel Mountains have been investigated. Coalification varies from 0,4 % Rr to 6,2 % Rmax and allows a significant description of the low diagenetic temperature distribution in the greatest part of this area. For most stratigraphiC units, reliable correlations between vitrinite reflectance and illite crystallinity can be established. By means of both methods the fields of diagenesis and anchimetamorphism are subdivided into five stages.A comprehensive coal rank map, based on outcrop specimen is published. It represents the amount and the distribution of the weak alpidic temperature effects within the uppermost Austroalpine nappes. From the northern to the southern rim of the Northern Calcareous Alps and in each tectonic unit, a general temperature increase is charateristic. Nevertheless, rank disconformities and anomalies are widespread, caused by tectonic structures. They depend on the pre-orogenetic increase in diagenesis with depth, preserved in every single nappe sheet. The anchimetamorphic stage appears at the southern rim of the Northern Calcareous Alps (Lechtal Unit) and was obviously developed during nappe-piling, i.e. synorogenctic (Eo-Alpidic). In the southwestern part of the Lechtal Alps a younger post-nappe distinctly minor overprint of maturity is found. It is attributed to the heating effect of the Tertiary (Meso- to Neo-Alpidic) metamorphism of the Penninic. A subsequent northward movement of the Ötzal-Stubai Complex, overriding the Northern Calcareous Alps, is proved by the absence of anchimetamorphic rocks along the Inn valley 25 km in length.
