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Background: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133+ and mesenchymal CD271+ stem cells was the object of the present study. Methods and Results: The effects of respective CD133+ and CD271+ mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271+ stem cells when compared with single CD133+ culture. Interestingly, CD133+ cells contributed in the tube formation, only if they were cultivated in combination with CD271+ cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor βeta 3 (TGFβ3) as well as an increase of the proangiogenic factors after CD133+ cell treatment in contrast to CD271+ cell treatment. On the other hand, the CD271+ cell therapy led to a lower expression of the inflammatory cytokines. Conclusion: The interactions between CD271+ and CD133+ subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271+ cells alone as well as in combination with CD133+ will result in an improved therapeutic impact on ischemic heart disease. [ABSTRACT FROM AUTHOR]

Background: CD133+ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133+ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics. Methods: CD133+ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133+ cells was evaluated and compared to manually isolated CD133+ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction. Results: We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 106 viable CD133+ cells with a mean log10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo. Conclusions: Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133+ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs. [ABSTRACT FROM AUTHOR]

Background: Sauna bathing is claimed to provide benefits for patients suffering from cardiovascular diseases. The current study aims at analyzing the induction of potential regenerative processes by quantifying the mobilization of bone marrow-derived stem cells into the peripheral blood of healthy adults following Finnish sauna. Materials and Methods: Twenty healthy unbiased male volunteers (20-30 years old) were exposed to a Finnish sauna bath (3 × 10 min, 90°C). Venous blood samples were drawn before (baseline), immediately, and 6 h as well as 24 h after the sauna bath. Blood analysis included isolation of mononuclear cells, cell staining with mononuclear antibodies, and fluorescence-activated cell sorting (FACS). For baseline and 24 h post-sauna samples colony-forming unit-Hill assays were applied to quantify endothelial progenitor cells (EPC). Results: Flow cytometry revealed an upregulation of circulating CD45+/CD309+ progenitor cells immediately after the sauna bath, however without reaching statistical significance. Circulating cell numbers of the CD45+CD34+, CD45+CD34+CD133+, and CD45+CD34+CD117+ populations did not show clear enhancements following sauna. EPC colony formation tended to be enhanced after sauna as compared to baseline values. Conclusion: Peripheral EPC numbers exhibited a moderate increase following Finnish sauna in a cohort of healthy young men. Furthermore, sauna bathing tended to increase EPC colony-forming capacity. These rather weak responses to thermotherapy might indicate a ceiling effect. In individuals exhibiting cardiovascular risk factors the effects may be more pronounced. © 2015 S. Karger GmbH, Freiburg [ABSTRACT FROM AUTHOR]

Myocardial infarction ( MI) is a major condition causing heart failure ( HF). After MI, the renin angiotensin system ( RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors ( AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4+ AT2R+ cells in the rat heart and spleen post-infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4+ AT2R+ T cells in circulating blood, post-infarcted heart and spleen represented 3.8 ± 0.4%, 23.2 ± 2.7% and 22.6 ± 2.6% of the CD4+ cells. CD4+ AT2R+ T cells within blood CD4+ T cells were reduced from 2.6 ± 0.2% in healthy controls to 1.7 ± 0.4% in patients. Moreover, we characterized CD4+ AT2R+ T cells which expressed regulatory FoxP3, secreted interleukin-10 and other inflammatory-related cytokines. Furthermore, intramyocardial injection of MI-induced splenic CD4+ AT2R+ T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4+ AT2R+ cells as a T cell subset improving heart function post- MI corresponding with reduced infarction size in a rat MI-model. Our results indicate CD4+ AT2R+ cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof. [ABSTRACT FROM AUTHOR]

Genetic modifications of bone marrow derived human mesenchymal stemcells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag).We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allow for initial cell modulation in vitro. [ABSTRACT FROM AUTHOR]

Background: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133 + and mesenchymal CD271 + stem cells was the object of the present study., Methods and Results: The effects of respective CD133 + and CD271 + mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271 + stem cells when compared with single CD133 + culture. Interestingly, CD133 + cells contributed in the tube formation, only if they were cultivated in combination with CD271 + cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor βeta 3 (TGFβ3) as well as an increase of the proangiogenic factors after CD133 + cell treatment in contrast to CD271 + cell treatment. On the other hand, the CD271 + cell therapy led to a lower expression of the inflammatory cytokines., Conclusion: The interactions between CD271 + and CD133 + subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271 + cells alone as well as in combination with CD133 + will result in an improved therapeutic impact on ischemic heart disease.