Claus Sondergaard

Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF‐BB), transforming growth factor β1 (TGF‐β1), and vascular endothelial growth factor (VEGF), in human bone marrow‐derived MSCs. Ectopic expression of bFGF or PDGF‐B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF‐B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF‐β1 blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin‐1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation. STEM CELLS 2011;29:1727–1737

Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction

Human stem cells from adult sources have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature. The mechanisms by which this is accomplished are, however, still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDHhiLin-, and ALDHloLin- cells following transplantation to NOD/SCID or NOD/SCID β2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDHhiLin- stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDHloLin- committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was, however, a significant increase in vascular density in the central infarct zone of ALDHhiLin- cell-treated mice, as compared to PBS and ALDHloLin- cell-treated mice.ConclusionsOur data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.

Mesenchymal Stem Cell Seeding of Porcine Small Intestinal Submucosal Extracellular Matrix for Cardiovascular Applications

In this study, we investigate the translational potential of a novel combined construct using an FDA-approved decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) seeded with human or porcine mesenchymal stem cells (MSCs) for cardiovascular indications. With the emerging success of individual component in various clinical applications, the combination of SIS-ECM with MSCs could provide additional therapeutic potential compared to individual components alone for cardiovascular repair. We tested the in vitro effects of MSC-seeding on SIS-ECM on resultant construct structure/function properties and MSC phenotypes. Additionally, we evaluated the ability of porcine MSCs to modulate recipient graft-specific response towards SIS-ECM in a porcine cardiac patch in vivo model. Specifically, we determined: 1) in vitro loading-capacity of human MSCs on SIS-ECM, 2) effect of cell seeding on SIS-ECM structure, compositions and mechanical properties, 3) effect of SIS-ECM seeding on human MSC phenotypes and differentiation potential, and 4) optimal orientation and dose of porcine MSCs seeded SIS-ECM for an in vivo cardiac application. In this study, histological structure, biochemical compositions and mechanical properties of the FDA-approved SIS-ECM biomaterial were retained following MSCs repopulation in vitro. Similarly, the cellular phenotypes and differentiation potential of MSCs were preserved following seeding on SIS-ECM. In a porcine in vivo patch study, the presence of porcine MSCs on SIS-ECM significantly reduced adaptive T cell response regardless of cell dose and orientation compared to SIS-ECM alone. These findings substantiate the clinical translational potential of combined SIS-ECM seeded with MSCs as a promising therapeutic candidate for cardiac applications.

Contractile and Electrophysiologic Characterization of Optimized Self-Organizing Engineered Heart Tissue

BACKGROUND Engineered heart tissue (EHT) is being developed for clinical implantation in heart failure or congenital heart disease and therefore requires a comprehensive functional characterization and scale-up of EHT. Here we explored the effects of scale-up of self-organizing EHT and present detailed electrophysiologic and contractile functional characterization. METHODS Fibers from EHT were generated from self-organizing neonatal rat cardiac cells (0.5×10(6) to 3×10(6)/fiber) on fibrin. We characterized contractile patterns and measured contractile function using a force transducer, and assessed force-length relationship, maximal force generation, and rate of force generation. Action potential and conduction velocity of EHT were measured with optical mapping, and transcript levels of myosin heavy chain beta were measured by reverse transcriptase-polymerase chain reaction. RESULTS Increasing the cell number per construct resulted in an increase in fiber volume. The force-length relationship was negatively impacted by increasing cell number. Maximal force generation and rate of force generation were also abrogated with increasing cell number. This decrease was not likely attributable to a selective expansion of noncontractile cells as myosin heavy chain beta levels were stable. Irregular contractile behavior was more prevalent in constructs with more cells. Engineered heart tissue (1×10(6)/construct) had an action potential duration of 140.2 milliseconds and a conduction velocity of 23.2 cm/s. CONCLUSIONS Engineered heart tissue displays physiologically relevant features shared with native myocardium. Engineered heart tissue scale-up by increasing cell number abrogates contractile function, possibly as a result of suboptimal cardiomyocyte performance in the absence of vasculature. Finally, conduction velocity approaches that of native myocardium without any electrical or mechanical conditioning, suggesting that the self-organizing method may be superior to other rigid scaffold-based EHT.

Lysophosphatidic Acid Enhances Stromal Cell-Directed Angiogenesis

Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and in severe cases result in ulcers, necrosis, and limb loss. While the therapeutic delivery of growth factors to promote angiogenesis has been widely investigated, large-scale implementation is limited by strategies to effectively deliver costly recombinant proteins. Multipotent adipose-derived stromal cells (ASC) and progenitor cells from other tissue compartments secrete bioactive concentrations of angiogenic molecules, making cell-based strategies for in situ delivery of angiogenic cytokines an exciting alternative to the use of recombinant proteins. Here, we show that the phospholipid lysophosphatidic acid (LPA) synergistically improves the proangiogenic effects of ASC in ischemia. We found that LPA upregulates angiogenic growth factor production by ASC under two- and three-dimensional in vitro models of serum deprivation and hypoxia (SD/H), and that these factors significantly enhance endothelial cell migration. The concurrent delivery of LPA and ASC in fibrin gels significantly improves vascularization in a murine critical hindlimb ischemia model compared to LPA or ASC alone, thus exhibiting the translational potential of this method. Furthermore, these results are achieved using an inexpensive lipid molecule, which is orders-of-magnitude less costly than recombinant growth factors that are under investigation for similar use. Our results demonstrate a novel strategy for enhancing cell-based strategies for therapeutic angiogenesis, with significant applications for treating ischemic diseases.

Human thymus mesenchymal stromal cells augment force production in self-organized cardiac tissue

BACKGROUND Mesenchymal stromal cells have been recently isolated from thymus gland tissue discarded after surgical procedures. The role of this novel cell type in heart regeneration has yet to be defined. The purpose of this study was to evaluate the therapeutic potential of human thymus-derived mesenchymal stromal cells using self-organized cardiac tissue as an in vitro platform for quantitative assessment. METHODS Mesenchymal stromal cells were isolated from discarded thymus tissue from neonates undergoing heart surgery and were incubated in differentiation media to demonstrate multipotency. Neonatal rat cardiomyocytes self-organized into cardiac tissue fibers in a custom culture dish either alone or in combination with varying numbers of mesenchymal stromal cells. A transducer measured force generated by spontaneously contracting self-organized cardiac tissue fibers. Work and power outputs were calculated from force tracings. Immunofluorescence was performed to determine the fate of the thymus-derived mesenchymal stromal cells. RESULTS Mesenchymal stromal cells were successfully isolated from discarded thymus tissue. After incubation in differentiation media, mesenchymal stromal cells attained the expected phenotypes. Although mesenchymal stromal cells did not differentiate into mature cardiomyocytes, addition of these cells increased the rate of fiber formation, force production, and work and power outputs. Self-organized cardiac tissue containing mesenchymal stromal cells acquired a defined microscopic architecture. CONCLUSIONS Discarded thymus tissue contains mesenchymal stromal cells, which can augment force production and work and power outputs of self-organized cardiac tissue fibers by several-fold. These findings indicate the potential utility of mesenchymal stromal cells in treating heart failure.

Minimal engraftment of human CD34+ cells mobilized from healthy donors in the infarcted heart of athymic nude rats.

Cell-based regenerative therapy may be useful for treatment of acute myocardial infarction (AMI). Animal xenograft models are ideally suited for preclinical studies evaluating prospective treatment regimes, identifying candidate human cell populations, and gaining mechanistic insight. Here we address whether the athymic nude rat is suitable as a xenograft model for the study of human CD34+ mobilized peripheral blood stem cells (M-PBSCs) in the repair of AMI. We injected human donor cells into the infarct border of athymic nude rats with surgically induced AMI and evaluated engraftment and functional improvement. We found no human engraftment by immunofluorescence staining at 14 days after transplantation or functional improvement at days 2 and 14 compared to controls. The lack of long-term human engraftment was furthermore confirmed in a time series study analyzing animals at 0, 24, 48, 72, and 96 h after transplantation. Although we found fluorescent microbeads coinjected with human CD34+ M-PBSCs at all time points, the number of donor cells rapidly declined and became undetectable at 96 h. CD34+ M-PBSCs from the same donor used to treat athymic nude rat hearts engrafted the bone marrow of nonobese diabetic/severe combined immunodeficient mice 8-10 weeks after transplantation. In conclusion, human CD34+ M-PBSCs with confirmed hematopoietic engraftment potential rapidly disappeared from the site of injury following intramyocardial transplantation in the athymic nude rat AMI model.

Cardiac tissue engineering

Publisher Summary Heart disease remains the leading cause of death and disability in the industrialized nations. The most frequent initiating cause of heart failure is myocardial infarction, also known as heart attack, which is the single most common cause of death in economically developed countries, including the US and Western Europe. Myocardial infarction typically results in fibrous (collagen) scar formation and permanently impaired cardiac function because, after a massive cell loss due to ischaemia, the myocardial tissue lacks significant intrinsic regenerative capability. Eventually, heart transplantation is the ultimate treatment option to end-stage heart failure. Owing to the lack of organ donors and complications associated with immune suppressive treatments, scientists and surgeons constantly look for new strategies to repair the injured heart. This chapter presents a review on the achievements of myocardial tissue engineering, focusing on construct (scaffold)-based strategies. A number of excellent reviews focusing on different aspects of cardiac tissue engineering, including cell-based therapies for myocardial regeneration, have been published recently.

Preloading Potential of Retroviral Vectors Is Packaging Cell Clone Dependent and Centrifugation onto CH-296 Ensures Highest Transduction Efficiency

Retroviral vector-mediated gene transfer has been used successfully in clinical gene therapy. Cells of the hematopoietic lineages, however, remain difficult to transduce, although precoating of culture vessels with the fibronectin fragment CH-296 may improve transduction efficiency. Alternatively, low-speed centrifugation of vector-containing supernatant onto culture vessels may improve transduction efficiency in the absence of CH-296 preloading. Using the NIH/3T3-derived Moloney murine leukemia virus-based packaging cell lines PG13, PA317, and PT67, we here show that preloading by low-speed centrifugation improves transduction efficiency in a packaging cell subclone-dependent manner. Preloading by centrifugation, however, cannot generally replace CH-296 and we obtained the overall highest transduction levels when combining centrifugation and CH-296 precoating. We found, moreover, that the factor responsible for high susceptibility to preloading in our PG13-derived vector supernatant was transferable to a PA317-derived vector supernatant with low susceptibility to preloading. Furthermore, our PA317, PG13, and PT67 subclones shed into their supernatants variable amounts of fibronectin. This soluble fibronectin formed aggregates of various sizes and generated complexes with vector particles. The fibronectin-vector complexes readily sedimented onto culture vessels and copurified after fibronectin-specific affinity purification of vector-containing supernatants. Finally, vector supernatant from 293T cells, which barely produce fibronectin, was not susceptible to preloading. The susceptibility to preloading by centrifugation thus appears to be dependent both on the specific packaging cell line and on the association of vector particles and packaging cell-produced fibronectin. Rigorous screening of individual vector-containing supernatants is therefore required to identify optimal transduction conditions for retroviral gene transfer.

Computational Analysis of Contractility in Engineered Heart Tissue

Engineered heart tissue (EHT) is a potential therapy for heart failure and the basis of functional in vitro assays of novel cardiovascular treatments. Self-organizing EHT can be generated in fiber form, which makes the assessment of contractile function convenient with a force transducer. Contractile function is a key parameter of EHT performance. Analysis of EHT force data is often performed manually; however, this approach is time consuming, incomplete and subjective. Therefore, the purpose of this study was to develop a computer algorithm to efficiently and objectively analyze EHT force data. This algorithm incorporates data filtering, individual contraction detection and validation, inter/intracontractile analysis and intersample analysis. We found the algorithm to be accurate in contraction detection, validation and magnitude measurement as compared to human operators. The algorithm was efficient in processing hundreds of data acquisitions and was able to determine force-length curves, force-frequency relationships and compare various contractile parameters such as peak systolic force generation. We conclude that this computer algorithm is a key adjunct to the objective and efficient assessment of EHT contractile function.