Paula Müller

Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105+ hMSCs

Genetic modifications of bone marrow derived human mesenchymal stem cells (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 allowfor initial cell modulation in vitro.

Cardiac Cell Therapies for the Treatment of Acute Myocardial Infarction: A Meta-Analysis from Mouse Studies.

Aims: Stem cell-based regenerative therapies for the treatment of ischemic myocardium are currently a subject of intensive investigation. A variety of cell populations have been demonstrated to be safe and to exert some positive effects in human Phase I and II clinical trials, however conclusive evidence of efficacy is still lacking. While the relevance of animal models for appropriate pre-clinical safety and efficacy testing with regard to application in Phase III studies continues to increase, concerns have been expressed regarding the validity of the mouse model to predict clinical results. Against the background that hundreds of preclinical studies have assessed the efficacy of numerous kinds of cell preparations - including pluripotent stem cells - for cardiac repair, we undertook a systematic re-evaluation of data from the mouse model, which initially paved the way for the first clinical trials in this field. Methods and Results: A systematic literature screen was performed to identify publications reporting results of cardiac stem cell therapies for the treatment of myocardial ischemia in the mouse model. Only peer-reviewed and placebo-controlled studies using magnet resonance imaging (MRI) for left ventricular ejection fraction (LVEF) assessment were included. Experimental data from 21 studies involving 583 animals demonstrate a significant improvement in LVEF of 8.59%+/- 2.36; p=.012 (95% CI, 3.7–13.8) compared with control animals. Conclusion: The mouse is a valid model to evaluate the efficacy of cell-based advanced therapies for the treatment of ischemic myocardial damage. Further studies are required to understand the mechanisms underlying stem cell based improvement of cardiac function after ischemia.

Cardiac Function Improvement and Bone Marrow Response

Objective The phase III clinical trial PERFECT was designed to assess clinical safety and efficacy of intramyocardial CD133+ bone marrow stem cell treatment combined with CABG for induction of cardiac repair. Design Multicentre, double-blinded, randomised placebo controlled trial. Setting The study was conducted across six centres in Germany October 2009 through March 2016 and stopped due slow recruitment after positive interim analysis in March 2015. Participants Post-infarction patients with chronic ischemia and reduced LVEF (25–50%). Interventions: Eighty-two patients were randomised to two groups receiving intramyocardial application of 5 ml placebo or a suspension of 0.5–5 × 106 CD133+. Outcome Primary endpoint was delta (∆) LVEF at 180 days (d) compared to baseline measured in MRI. Findings (prespecified) Safety (n = 77): 180 d survival was 100%, MACE n = 2, SAE n = 49, without difference between placebo and CD133+. Efficacy (n = 58): The LVEF improved from baseline LVEF 33.5% by + 9.6% at 180 d, p = 0.001 (n = 58). Treatment groups were not different in ∆ LVEF (ANCOVA: Placebo + 8.8% vs. CD133+ + 10.4%, ∆ CD133+ vs placebo + 2.6%, p = 0.4). Findings (post hoc) Responders (R) classified by ∆ LVEF ≥ 5% after 180 d were 60% of the patients (35/58) in both treatment groups. ∆ LVEF in ANCOVA was + 17.1% in (R) vs. non-responders (NR) (∆ LVEF 0%, n = 23). NR were characterized by a preoperative response signature in peripheral blood with reduced CD133+ EPC (RvsNR: p = 0.005) and thrombocytes (p = 0.004) in contrast to increased Erythropoeitin (p = 0.02), and SH2B3 mRNA expression (p = 0.073). Actuarial computed mean survival time was 76.9 ± 3.32 months (R) vs. + 72.3 ± 5.0 months (NR), HR 0.3 [Cl 0.07–1.2]; p = 0.067.Using a machine learning 20 biomarker response parameters were identified allowing preoperative discrimination with an accuracy of 80% (R) and 84% (NR) after 10-fold cross-validation. Interpretation The PERFECT trial analysis demonstrates that the regulation of induced cardiac repair is linked to the circulating pool of CD133 + EPC and thrombocytes, associated with SH2B3 gene expression. Based on these findings, responders to cardiac functional improvement may be identified by a peripheral blood biomarker signature. TRIAL REGISTRATION: ClinicalTrials.govNCT00950274.

Magnet-Bead Based MicroRNA Delivery System to Modify CD133(+) Stem Cells.

Aim. CD133+ stem cells bear huge potential for regenerative medicine. However, low retention in the injured tissue and massive cell death reduce beneficial effects. In order to address these issues, we intended to develop a nonviral system for appropriate cell engineering. Materials and Methods. Modification of human CD133+ stem cells with magnetic polyplexes carrying microRNA was studied in terms of efficiency, safety, and targeting potential. Results. High microRNA uptake rates (~80–90%) were achieved without affecting CD133+ stem cell properties. Modified cells can be magnetically guided. Conclusion. We developed a safe and efficient protocol for CD133+ stem cell modification. Our work may become a basis to improve stem cell therapeutical effects as well as their monitoring with magnetic resonance imaging.

Data on the fate of MACS® MicroBeads intramyocardially co-injected with stem cell products

The data presented in this article are related to the research article “Intramyocardial Fate and Effect of Iron Nanoparticles co-injected with MACS® purified Stem Cell Products” (Müller et al., 2017) [1]. This article complements the cellular localization of superparamagnetic iron dextran particles (MACS® MicroBeads) used for magnetic activated cell sorting (MACS®). Data evaluate the time-dependent detachment of these nanoparticles from CD133+ haematopoietic stem cells (HSCs) and CD271+ mesenchymal stem cells (MSCs). Furthermore, the influence of these stem cells as well as of nanoparticles on cardiac remodeling processes after myocardial infarction (MI) was investigated.

Stem cells and heart disease - Brake or accelerator?

After two decades of intensive research and attempts of clinical translation, stem cell based therapies for cardiac diseases are not getting closer to clinical success. This review tries to unravel the obstacles and focuses on underlying mechanisms as the target for regenerative therapies. At present, the principal outcome in clinical therapy does not reflect experimental evidence. It seems that the scientific obstacle is a lack of integration of knowledge from tissue repair and disease mechanisms. Recent insights from clinical trials delineate mechanisms of stem cell dysfunction and gene defects in repair mechanisms as cause of atherosclerosis and heart disease. These findings require a redirection of current practice of stem cell therapy and a reset using more detailed analysis of stem cell function interfering with disease mechanisms. To accelerate scientific development the authors suggest intensifying unified computational data analysis and shared data knowledge by using open-access data platforms.

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80-90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

Stem Cell Therapy in Heart Diseases – Cell Types, Mechanisms and Improvement Strategies

A large number of clinical trials have shown stem cell therapy to be a promising therapeutic approach for the treatment of cardiovascular diseases. Since the first transplantation into human patients, several stem cell types have been applied in this field, including bone marrow derived stem cells, cardiac progenitors as well as embryonic stem cells and their derivatives. However, results obtained from clinical studies are inconsistent and stem cell-based improvement of heart performance and cardiac remodeling was found to be quite limited. In order to optimize stem cell efficiency, it is crucial to elucidate the underlying mechanisms mediating the beneficial effects of stem cell transplantation. Based on these mechanisms, researchers have developed different improvement strategies to boost the potency of stem cell repair and to generate the “next generation” of stem cell therapeutics. Moreover, since cardiovascular diseases are complex disorders including several disease patterns and pathologic mechanisms it may be difficult to provide a uniform therapeutic intervention for all subgroups of patients. Therefore, future strategies should aim at more personalized SC therapies in which individual disease parameters influence the selection of optimal cell type, dosage and delivery approach.