Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-β mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.

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We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery.

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In regenerative medicine, there is an approach to avoid expansion of the mesenchymal stem cell (MSC) before implantation. The aim of this study was to compare methods for instant MSC therapy by use of a portable, automatic and closed system centrifuge that allows for the concentration of MSCs. The main outcome measures were the amount of MSCs per millilitre of bone marrow (BM), clusters of differentiation (CD), proliferation and differentiation capacities of the MSC. A volume reduction protocol was compared to the traditional laboratory methods of isolation using a Ficoll gradient and native BM. Fifty millilitres of BM were obtained from haematologically healthy male Caucasians (n= 10, age 8 to 49 years). The number of colony forming units-fibroblast (CFU-F)/ml BM was highest in the centrifuge volume reduction protocol, followed by the native BM (not significant), the centrifuge Ficoll (p= 0.042) and the manual Ficoll procedure (p=0.001). The MSC of all groups could differentiate into the mesenchymal lineages without significant differences between the groups. The CD pattern was identical for all groups: CD13+; CD 44+; CD73 +; CD90+; CD105+; HLA-A,B,C+; CD14-; CD34-; CD45-; CD271-; HLA-DR-. In a further clinical pilot study (n=5) with 297 ml BM (SD 18.6), the volume reduction protocol concentrated the MSC by a factor of 14: there were 1.08 x 10 2 MSC/ml BM (standard deviation (SD) 1.02 x 10 2) before concentration, 14.8 x 10 2 MSC/ ml BM (SD 12.4 x 10 2) after concentration, and on average 296 x 10 2 MSC (SD 248.9 x 10 2 , range 86.4-691.5 x 10 2) were available for MSC therapy. The volume reduction protocol of the closed centrifuge allows for the highest concentration of the MSC, and therefore, is a promising candidate for instant stem cell therapy.