The transplantation of BMCs as well as TRCs proved to be safe and feasible. Improvements of microcirculation and complete wound healing were observed in the transplant groups.

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insuffi cient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the fi rst time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profi le and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated effi ciently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defi ned medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Heparin-induced thrombocytopenia was more prevalent in patients receiving mechanical circulatory support than in other cardiac patients. Frequent antibody screening is recommended due to the increased risk of thromboembolism. Heparin alternatives should be subjected to clinical trials in these high-risk patients.

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105 + CD166 + CD14 − CD45 − and fibroblastic CFU, expanded 317-and 364-fold, respectively, while CD34 + hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 × 10 6 total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

IgG classification of anti-PF4/heparin antibodies to identify patients with heparin-induced thrombocytopenia during mechanical circulatory support. See also Warkentin TE. PF4-dependent immunoassays and inferential detection of HIT antibodies. This issue, pp 232-4.Summary. Commercial immunoassays frequently detect anti-PF4/heparin antibodies during mechanical circulatory support (MCS), but only a small minority of patients develops heparininduced thrombocytopenia (HIT). Whereas platelet functional tests can distinguish between platelet-activating and nonplatelet-activating antibodies, commercial PF4-dependent immunoassays do not. Between 2003 and 2004, 113 patients were placed on MCS. Blood samples were obtained on postimplant day 5-7 for analyses by antibody assays and the functional heparin-induced platelet activation (HIPA) assay. Three distinct groups of patient sera were identified: platelet-activating anti-PF4/heparin antibodies (n ¼ 10), non-platelet-activating anti-PF4/heparin antibodies (n ¼ 53), and anti-PF4/ heparin antibody negative (n ¼ 50). Patients with plateletactivating antibodies had the highest risk for thromboembolic events (P < 0.005), whereas those with non-platelet-activating antibodies did not differ from antibody negative patients (P ¼ 0.369). The enzyme-immunoassay and column agglutination assays, which cover all immunoglobulin classes, demonstrated adequate sensitivity and negative predictive value; yet, both lacked specificity with respect to the platelet-activating antibodies. If all antibody positive patients were further classified by an IgG-specific anti-PF4/heparin enzyme-immuno assay, specificity for platelet-activating antibodies increased. Whereas IgG-specific optical density (OD) values below 1.0 were likely for non-platelet-activating anti-PF4/heparin antibodies, higher values were progressively predictive for pathogenic platelet activation. The probability of the development of clinical HIT also increased steeply. In conclusion, platelet-activating anti-PF4/heparin antibodies are relatively common (about 9%) in patients on MCS and are associated with significantly higher thrombotic event rates. Low IgG-specific OD values (< 1.0) in the enzyme-immunoassay indicate low likelihood for the presence of platelet-activating antibodies. These results justify further validation so that anticoagulation during MCS becomes safer and adequate.

Previous studies suggest that autologous transplantation of bone marrow mononuclear cells is safe and effective in inducing therapeutic angiogenesis in patients with peripheral arterial occlusive disease (PAOD). Here we discuss a multidisciplinary approach to treating PAOD with a focus on the use of angiological diagnostic tools. We conclude that our autologous stem cell therapy is working in this patient and it is a potential new therapeutic option for diabetic patients with chronic foot ulcers induced by critical limb ischaemia.

Summary:The synthesis of tissue factor pathway inhibitor (TFPI) was investigated in cloned human synovial cells and human chondrocytes. TFPI-specific DNA transcription products of these cells were isolated, and a full-length cDNA of about 1000 base pairs was amplified by reverse transcription and polymerase chain reaction. The amplified DNA was cloned into the vector pUC 18. The TFPI coding sequence was confirmed by double stranded sequencing and was identical with that previously published for human TFPI coding nucleotide sequence frorn human placental cDNA (1).The inhibitory activity of TFPI in the cell medium of cultivated human chondrocytes and cloned human synovial cells was determined by a specific chromogenic Substrate assay of factor Xa activity. The inhibitory activity of TFPI in the medium of human chondrocytes and cloned human synovial cells was 630-720 mU/10 8 cells and 1080-1665 mU/10 8 cells, respectively.In addition, TFPI activity in cell culture media of human chondrocytes and cloned human synovial cells was suppressed by a polyclonal goat anti-TFPI antibody directed against the inhibitory domain I and domain II. In the chromogenic Substrate assay, the anti-TFPl antibody completely suppressed the inhibitory activity of TFPI in the samples.

The aim of our study was to determine the prevalence of the factor V mutation (position 1691 G-->A) in patients with angiographically diagnosed coronary artery disease and myocardial infarction and, as a control, in blood donors. This mutation has already been proved to be the main genetic risk factor for venous thrombosis. In order to detect this mutation in exon 10 of the factor V gene we established a microtiter plate based hybridization assay for the specific detection of wild-type and mutant sequences in factor V gene segments, obtained after amplification by polymerase chain reaction. This test enables us to screen a large number of samples. The mutation was detected in 29 of 317 coronary artery disease (CAD) patients (9.1%) and 18 of 190 blood donors (9.5%) investigated. The mean activated protein C resistance ratios were 3.18 and 3.11, with nearly identical distribution. No increased prevalence of the factor V mutation was found in the CAD group. In 10 of 29 CAD patients (35%) with the factor V 1691 G-->A mutation and in 124 of 288 CAD patients without the mutation (43%) there was a history of myocardial infarction. From our data we conclude that there is no increased risk of developing coronary atheroma or consecutive myocardial infarction resulting from the factor V mutation with protein C resistance.