GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration.

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.

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.

The CD4(+) AT2R(+) T cell subpopulation improves post-infarction remodelling and restores cardiac function.

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.

Impact of short-term liquid storage on human CD133(+) stem cells.

Stem cell transplantation is a viable strategy for regenerative medicine. However, it is inevitable to have cells undergo storage for several hours or days due to processing and transportation. Therefore, it is crucial to have rigidly controlled conditions ensuring the therapeutic benefit of isolated stem cells. In the present study, we investigated the impact of short-term storage on human CD133(+) cells. CD133(+) cells were isolated from human bone marrow and kept at standardized nonfreezing storage conditions for up to 72 h. Cell viability (apoptosis/necrosis) and expression of CD133 and CXCR4 were analyzed by flow cytometry. Metabolic activity was determined using an MTT assay; colony-forming ability, as well as endothelial-like differentiation, was further evaluated. A qRT-PCR array was employed to investigate the expression of stemness genes. CD133 and CXCR4 expressions were preserved at all time points. After 30 h, cell number and metabolic activity decreased, although no significant changes were detected in cell viability and proliferation as well as endothelial-like differentiation. Cell viability and proliferation decreased significantly only after 72 h of storage. Our results indicate that storage of isolated human CD133(+) bone marrow stem cells in liquid allows for high viability and functionality. However, storage time should be limited in order to avoid cell loss.

Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient.

Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.

Human Mesenchymal Stem Cells Display Reduced Expression of CD105 after Culture in Serum-Free Medium.

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However, ex vivo expansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS.

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL-6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well-studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll-like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down-regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up-regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS-treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin-6 (IL-6), a potent stimulator of cell migration. Interestingly, the levels of IL-6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental-derived progenitor cells in sites of periodontal infection.

Analyzing migratory properties of human CD133(+) stem cells in vivo after intraoperative sternal bone marrow isolation.

Human bone marrow stem cell populations have been applied for cardiac regeneration purposes within different clinical settings in the recent past. The migratory capacity of applied stem cell populations towards injured tissue, after undergoing specific peri-interventional harvesting and isolation procedures, represents a key factor limiting therapeutic efficacy. We therefore aimed at analyzing the migratory capacity of human cluster of differentiation (CD) 133(+) bone marrow stem cells in vivo after intraoperative harvesting from the sternal bone marrow. Human CD133(+) bone marrow stem cells were isolated from the sternal bone marrow of patients undergoing cardiac surgery at our institution. Migratory capacity towards stromal cell-derived factor-1α (SDF-1α) gradients was tested in vitro and in vivo by intravital fluoresecence microscopy, utilizing the cremaster muscle model in severe combined immunodeficient (SCID) mice and analyzing CD133(+) cell interaction with the local endothelium. Furthermore, the role of a local inflammatory stimulus for CD133(+) cell interaction with the endothelium was studied. In order to describe endothelial response upon chemokine stimulation laser scanning microscopy of histological cremaster muscle samples was performed. SDF-1α alone was capable to induce relevant early CD133(+) cell interaction with the endothelium, indicated by the percentage of rolling CD133(+) cells (45.9±1.8% in "SDF-1" vs. 17.7±2.7% in "control," p<0.001) and the significantly reduced rolling velocity after SDF-1α treatment. Furthermore, SDF-1α induced firm endothelial adhesion of CD133(+) cells in vivo. Firm endothelial adhesion, however, was significantly enhanced by additional inflammatory stimulation with tumor necrosis factor-α (TNF-α) (27.9±4.3 cells/mm(2)in "SDF-1 + TNF" vs. 2.2±1.1 cells/mm(2) in "control," p<0.001). CD133(+) bone marrow stem cells exhibit sufficient in vivo homing towards SDF-1α gradients in an inflammatory microenvironment after undergoing standardized intraoperative harvesting and isolation from the sternal bone marrow.

Polyethylenimine-mediated gene delivery into human bone marrow mesenchymal stem cells from patients.

Transplantation of mesenchymal stem cells (MSCs) derived from adult bone marrow has been proposed as a potential therapeutic approach for post-infarction left ventricular (LV) dysfunction. However, age-related functional decline of stem cells has restricted their clinical benefits after transplantation into the infarcted myocardium. The limitations imposed on patient cells could be addressed by genetic modification of stem cells. This study was designed to improve our understanding of genetic modification of human bone marrow derived mesenchymal stem cells (hMSCs) by polyethylenimine (PEI, branched with Mw 25 kD), one of non-viral vectors that show promise in stem cell genetic modification, in the context of cardiac regeneration for patients. We optimized the PEI-mediated reporter gene transfection into hMSCs, evaluated whether transfection efficiency is associated with gender or age of the cell donors, analysed the influence of cell cycle on transfection and investigated the transfer of therapeutic vascular endothelial growth factor gene (VEGF). hMSCs were isolated from patients with cardiovascular disease aged from 41 to 85 years. Optimization of gene delivery to hMSCs was carried out based on the particle size of the PEI/DNA complexes, N/P ratio of complexes, DNA dosage and cell viability. The highest efficiency with the cell viability near 60% was achieved at N/P ratio 2 and 6.0 µg DNA/cm(2) . The average transfection efficiency for all tested samples, middle-age group (<65 years), old-age group (>65 years), female group and male group was 4.32%, 3.85%, 4.52%, 4.14% and 4.38%, respectively. The transfection efficiency did not show any correlation either with the age or the gender of the donors. Statistically, there were two subpopulations in the donors; and transfection efficiency in each subpopulation was linearly related to the cell percentage in S phase. No significant phenotypic differences were observed between these two subpopulations. Furthermore, PEI-mediated therapeutic gene VEGF transfer could significantly enhance the expression level.

Potential protective role of apoprotein J (clusterin) in atherogenesis: binding to enzymatically modified low-density lipoprotein reduces fatty acid-mediated cytotoxicity.

Following entrapment in the arterial intima, low-density lipoprotein (LDL) can be modified by hydrolytic enzymes to yield a lipoprotein derivative that binds C-reactive protein, activates complement, and is rapidly taken up by monocytes/macrophages. Free fatty acids contained in enzymatically modified LDL (E-LDL) render the lipoprotein cytotoxic due to their capacity to trigger programmed cell death. Apoprotein J (ApoJ) alias clusterin is a multifunctional glycoprotein with cytoprotective and anti-inflammatory properties. It interacts with diverse substrates, is present in the intima and the media of arteries with atherosclerotic lesions and is also synthesized by smooth muscle cells during development of atherosclerosis. We report that ApoJ binds to E-LDL but not to native LDL. Binding resulted in marked reduction of cytotoxicity of E-LDL on smooth muscle cells, as revealed by determination of caspase activity, annexin binding, and cellular ATP. ApoJ was detected immunohistochemically in early atherosclerotic lesions, where it was found to co-localize with E-LDL. In atherosclerotic lesions, ApoJ may thus subserve protective functions through its capacity to inactivate C5b-9 complement complexes and by reducing the cytotoxic effects of modified LDL on cells that gain contact with the lipoprotein.