Keynote Lecture

Current approaches to the regeneration of bone tissue lack the appropriate support structure necessary to regain full function quickly. Today clinical solutions to bone loss often rely on a strong metallic alloy support. For example, large bone defects such as those caused by bone cancer utilize either large allografts supported by plates and screws, or massive metallic prostheses. These tumors occur in young patients and regenerative solutions to replace the defect and enable continued skeletal growth are a long way off. Therefore, mechanical solutions are still required. However, intermediate approaches where tissue engineering scaffolds are combined with structural supports such as allografts or metal implants are close to being used.

One such approach is to augment bone regeneration during surgery using stem cells sprayed onto the surface of the implant. This has been shown to significantly increase fixation of implants and implant longevity. Larger defects caused either by a disease process or by osteolysis owing to a previous implant can be effectively regenerated using a tissue engineering approach composed of scaffolds and stem cells. To do this the stem cells have to survive the mechanical forces acting on the scaffolds during insertion and use. These forces need to be measured and the cells used have to be able to withstand these high forces whilst being able to proliferate and differentiate along an osteoblastic lineage. The scaffolds used are generally granular and have to be impacted into place to fill the irregular-shaped defects. Cells in combination with impaction grafting do induce more bone formation and greater osteointegration of revision implants, resulting in improvement of joint function.

The structure and composition of the granules is important with some scaffolds being osteoinductive, even without the addition of growth factors or stem cells. The property that gives them their osteoinductivity is associated with their chemistry and morphology. Small pores within the scaffold structure, less than 50 mm, are important for bone formation in ectopic sites. The size of these pores is well below the optimal size for ingrowth of bone into porous implant surfaces. The chemistry of the implant is also important. Bone formation within these scaffolds is further increased by the addition of stem and osteoprogenitor cells.

During embryonic development and embryonic stem cell (ESC) differentiation, the different cells forming the mature heart arise from the differentiation of two types of multipotent cardiovascular progenitors (MCPs) contributing to first and second heart fields. Using mouse eESC in which gene expression can be temporally regulated, we found that transient expression of Mesp1 dramatically accelerates and enhances MCP specification through an intrinsic and cell autonomous mechanism. Mesp1 is a key regulator of MCP specification that rapidly and directly activates the expression of a discrete set of genes belonging to the core cardiac transcriptional machinery, leading to MCP specification. Mesp1 also directly represses the expression of key genes regulating other early mesoderm and endoderm cell fates. Mesp1 expression is the earliest step of cardiovascular development and can be used to isolate early common MCPs for both heart fields, which differentiate into cardiomyocytes, endothelial and smooth muscle cells both in vitro and in vivo. Using transcriptional profiling of these early Mesp1-expressing MCPs, we identified a combination of cell surface markers allowing their isolation andcellular and molecular characterization. Several transcription factors including Isl1 are expressed together with Mesp1 during MCP specification and control defined aspects of MCP specification and early cardiovascular lineage commitment. Isl1 is expressed in a subset of early Mesp1-expressing cells independently of Mesp1 and acts together with Mesp1 to promote cardiovascular differentiation. Our study identifies the early MCPs residing at the top of the cellular hierarchy of cardiovascular lineages during ESC differentiation and uncovers novel regulatory mechanisms that govern MCP specification and early cardiovascular lineage commitment.

Disclosure

This work was supported by the Belgian National Fund for Scientific Research, by a career development award of the Human Frontier Science Program Organization (HFSPO), a research grant of the Schlumberger Foundation, the program CIBLES of the Wallonia Region, a research grant from the ‘Fondation Contre le Cancer’ and the ‘Fond Gaston Ithier’, a starting grant of the European Research Council (ERC) and the EMBO Young Investigator Program.

Tissue engineering holds great promise for advancing the field of cardiovascular surgery where complications arising from the use of currently used synthetic vascular grafts is a leading cause of postoperative morbidity and mortality. We developed the first tissue engineered vascular graft to be used in humans. Our tissue engineered vascular graft is created by seeding autologous bone marrow-derived mononuclear cells onto a biodegradable tubular scaffold. As the scaffold degrades, neotissue forms ultimately creating a neovessel that resembles a native blood vessel. We targeted the pediatric population and used the tissue engineered vascular graft as a conduit in children undergoing congenital heart surgery in order to take advantage of the growth potential of these living vascular conduits. Results of our initial pilot study demonstrated the feasibility of using this technology in humans and confirmed the growth potential of these conduits, making them the first man-made vascular grafts with growth potential. Results of this study also demonstrated that while the tissue engineered vascular graft has a reasonable safety profile, stenosis was the leading graft-related complication. We subsequently developed a murine model for investigating the process of neovessel formation and used it to investigate the cellular and molecular mechanisms of neovessel formation with special emphasis on processes that influence the development of tissue engineered vascular graft stenosis. To date, we have demonstrated that while the cells seeded onto the tissue engineered vascular graft are critical to the process of neovessel formation, they rapidly disappear after implantation. We have shown that the neovessel is formed from cells derived from the neighboring blood vessel wall and that the process of neovessel formation is immune mediated. In this session we will review the results of our clinical study, highlight our findings related to the cellular and molecular mechanisms underlying neovessel formation and finally discuss how we are using this information to develop methods for detecting, treating and preventing the formation of tissue engineered vascular graft stenosis.

The purpose of this lecture is to illustrate how translational cellular imaging is expected to play a key role in evaluating the outcome of many cellular regenerative medicine clinical trials. In order to facilitate and implement the translation of novel experimental cell therapies into the clinic, one needs to be able to monitor the cellular biodistribution noninvasively following administration. Among the different clinically used cellular imaging techniques, ¹¹¹In oxine scintigraphy is the only US FDA-approved method and has been primarily used for imaging of infection and inflammation. Cellular MRI, with its superior spatial resolution and excellent soft tissue anatomical detail, is emerging as the technique of choice to monitor in real time image-guided cell delivery, immediate engraftment and short-term homing. Up until last year, seven clinical studies had been published, all using superparamagnetic iron oxide nanoparticles in an off-label fashion. Superparamagnetic iron oxide nanoparticles are clinically approved and create strong local magnetic field disturbances that spoil the magnetic resonance signal leading to hypo- or hyperintense contrast. A major setback is that the particles that were being used have been taken off the market, as their primary, FDA-approved indication (liver imaging of Kupffer cells) did not live up to its promise. However, several companies have started production runs of novel particles that possibly can also be used for magnetic particle imaging. Several other cellular imaging techniques are available, some of which are based on reporter genes, for example firefly luciferase for bioluminescent imaging and herpes simplex virus thymidine kinase for PET imaging. While the former cannot be used clinically because of physico-optical constraints, the latter has now also entered the clinic.

Stem cells respond to many cues from their microenvironment, which may include chemical signals, mechanics and topography. Importantly, these cues may be incorporated into scaffolding to optimize the ability of stem cells to produce tissues in regenerative medicine. Our laboratory is particularly interested in the development of photopolymerizable hydrogels as synthetic microenvironments for the repair of musculoskeletal tissues using mesenchymal stem cells (MSCs). Photopolymerization is a process that has many applications in the fabrication of biomaterials for drug delivery, in the design of microdevices and especially for tissue engineering. Through the addition of a photoinitiator and an initiating light source, liquid solutions containing multifunctional monomers solidify into crosslinked networks. The widespread application of this process has been motivated by the spatial and temporal control that is afforded during photoinitiated polymerizations.

Towards cartilage regeneration, we have been designing hydrogels based on hyaluronic acid (HA) that interact with cells via surface receptors (e.g., CD44) and degrade via hyaluronidases. When MSCs are encapsulated in HA hydrogels, enhanced chondrogenesis (upregulation of type II collagen, aggrecan) is noted compared with inert hydrogels (e.g., polyethylene glycol) and chondrogenesis is observed even without growth factors present. Our recent efforts are towards HA hydrogels containing matrix metalloproteinases degradable units formed through Michael-type reactions, where cells readily spread when encapsulated as long as adhesion sites (i.e., RGD) are present. If the Michael-type reaction and photopolymerization are used sequentially, this spreading can be spatially controlled in the hydrogels, leading the way to more complex tissue structures and altering stem cell fate decisions. This same approach is useful to spatially and temporally control mechanical properties, which also influences stem cell interactions (e.g., proliferation and differentiation). Overall, these advanced HA hydrogels provide us the opportunity to investigate diverse and controlled material properties on MSC interactions.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cells generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar observations were made in hair follicles and stomach epithelium.

Single sorted Lgr5-positive stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5-positive stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a nonepithelial cellular niche. The same technology has now been developed for the Lgr5-positive stomach stem cells.

Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells, results in progressively growing neoplasia, identifying the stem cell as the cell of origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the cancer stem cell concept.

Fate mapping of individual crypt stem cells using a multicolor Cre reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1–6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or transit-amplifying fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24⁺ and express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem cell maintenance in culture. Coculturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24⁺ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Epithelial stem cells are responsible for the continuous renewal and repair of human stratified epithelia, are clonogenic and are known as holoclones. The stem cells of the human corneal epithelium are located in the limbus, the narrow zone between the cornea and the bulbar conjunctiva. Self-renewal and proliferation of limbal stem cells are regulated by the DNp63 (a, b and g), C/EBPd and Bmi1 transcription factors.

Ocular burns may destroy the limbus, causing limbal stem cell deficiency. In such cases, the cornea acquires an epithelium through the invasion of bulbar conjunctival cells. This process causes neovascularization, chronic inflammation and stromal scarring, leading to corneal opacity and loss of vision. Allogeneic corneal transplantation, aimed at replacing the scarred corneal stroma and the inner endothelium, is not in itself a successful treatment. Although it temporarily removes the opacity, the conjunctival cells will resurface the cornea. The only way to prevent this invasion is to restore the limbus. The finding that human limbal cell cultures contain stem cells (detected as holoclones) led to the first therapeutic use of such cultures in the regeneration of corneal epithelium.

Here we report long-term (up to 10 years) clinical results obtained in an homogeneous group of 112 patients presenting with corneal opacification and visual loss owing to chemical burn-dependent limbal stem cell deficiency (86.6% unilateral and 13.4% bilateral) and treated with autologous limbal stem cell cultures. Clinical results were assessed by means of Kaplan–Meier, Kruskal–Wallis, and univariate and multivariate logistic-regression analyses.

Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up (mean: 2.91 ± 1.99; median: 1.93). In post-hoc analyses, success was associated with the percentage of holoclone-forming stem cells in culture. Cultures in which stem cells (detected as p63-bright cells) constituted more than 3% of the total number of clonogenic cells were associated with the permanent regeneration of a functional corneal epithelium in 80% of patients. By contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 10% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications.

We also show that cultures established from a single 1–2 mm limbal biopsy offer an opportunity to treat virtually blind patients who have severe bilateral loss of corneal epithelium, provided that a tiny part of the limbus is spared in one of the two eyes.

Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation (SCT) and induced by donor T-cells recognizing major or minor histocompatibility antigens of the recipient. After their activation and expansion, such alloreactive effector T-cells attack typical target organs, such as the skin, liver and gut. The main goal of current research in SCT is the separation of beneficial donor T-cell effects, such as the graft-versus-leukemia/lymphoma response of donor T-cells, from harmful and potentially life-threatening effects, such as severe GVHD. In murine disease models, we previously showed that the adoptive transfer of donor CD4⁺CD25⁺ Treg cells does not induce GVHD after allogeneic SCT, but protects from GVHD otherwise induced by co-transplanted conventional donor T-cells [1]. Importantly, donor Treg cells do not completely paralyze donor T-cell functions, as their graft-versus leukemia/lymphoma activity can be maintained in the presence of Treg cells [2]. Thus, the adoptive transfer of CD4⁺CD25⁺ Treg cells seems an attractive strategy for the prevention of GVHD after allogeneic SCT in humans. For the preparation of such clinical trials we described methods for the GMP-compatible isolation and in-vitro expansion of human Treg cells [3,4]. Furthermore, we showed that only CD45RA⁺ Treg cells generate homogeneous Treg cell lines after in-vitro expansion [5], while even CD127-depleted CD4⁺CD25⁺ T-cells partially lose their Treg cell characteristics [6], as illustrated by the loss of FOXP3 expression, the emergence of cytokine producers and by changes in the methylation pattern within the FOXP3 locus [7]. Detailed analyses of Treg cells that lost FOXP3 expression upon expansion revealed a switch towards a Th2-like differentiation pattern. Based on these various findings, we suggested that the isolation and expansion of naive Treg cells is the safest strategy for clinical trials in SCT exploring the suppressive activity of in-vitro-expanded Treg cells for GVHD prevention or GVHD therapy. Strategies and preliminary results from the efforts to translate these findings into clinical trials will be presented in this session.

Regenerative medicine is an exciting new field where rapid scientific advances are being achieved, although the delivery of new therapies in the clinic has been much slower. When it comes to translating research from bench to bedside, many of the pioneering innovations are achieved by cooperating teams of basic scientists and clinicians. In veterinary medicine many of these new and promising treatment options have been more rapidly translated into routine clinical practice, which has enabled considerable experience to be gained from the treatment of naturally occurring, rather than experimentally induced, disease. This talk will give an overview of the progress in veterinary regenerative medicine and the current implementation into clinical practice. Furthermore, an outlook on the most promising future applications will be given.

By isolating the inner cell mass from day 7 equine pre-implantation blastocysts we have derived self-renewing, pluripotent embryo-derived stem cells (ESCs). When cultured in the presence of embryonic feeder cells, the equine ESCs grow as colonies that can be expanded extensively whilst retaining the expression of numerous ESC markers. The expression pattern of these markers in equine ESCs reflects their expression in the equine blastocysts from which the ESCs are derived. Equine ESCs can differentiate in vitro into endoderm, ectoderm and mesoderm derivatives, but they have not been shown to form teratomas following their injection into severe combined immunodeficient mice.

Objective

To determine if the extensive expansion capacity and ability to undergo multilineage differentiation of equine ESCs make them a suitable source of off-the-shelf cells for aiding tendon repair in the horse.

Materials & methods

Undifferentiated equine ESCs and mesenchymal stromal cells expressing different reporter genes were injected into experimentally induced tendon lesions in horses. Cell survival and distribution were examined up to 90 days postmortem and host immune responses determined. In vivo differentiation of the ESCs was assessed using immunocytochemistry for the tendon progenitor marker scleraxis and an in vitro model of tendon differentiation by ESCs has been established using 3D cultures.

Results & conclusion

We demonstrated that neither cell type produced a detectable cell mediated immune response when used allogeneically. However, whereas both autologous and allogeneic mesenchymal stromal cells had a very poor survival (<5% after 10 days and <0.1% after 90 days), ESCs survived at a high and consistent level (>60%) for the 90-day time period studied. No abnormal growths were detected within the experimental duration, and histological and ultrasonographical repair progressed normally. In vivo, the ESCs express scleraxis, suggesting that the cells may be undergoing differentiation to a tenocyte fate. Scleraxis is required for normal tendon development and is upregulated in response to TGF-β signaling and we have shown that both scleraxis and TGF-β levels are increased in the damaged horse tendon. Exposure of equine ESCs to TGF-βin vitro enhances the expression of scleraxis and other genes associated with tendon differentiation. Using 3D culture in combination with TGF-β signaling we have established an in vitro model of tendon differentiation by equine ESCs, which will be used to better define the signaling cascade that leads to functional tendon-gene expression and qualitatively determine if the cells produced are capable of de novo synthesis of matrix components and reorganizing an extracellular matrix.

The hedgehog (Hh) pathway has a pivotal function in development through modulation of stem cells (SCs). To identify the mediators of this function, we observed that the transcription factor Nanog, which controls stemness as a key determinant of both embryonic SC self-renewal and differentiated somatic cell reprogramming to pluripotency, acts as a critical mediator of Hh-driven self-renewal, through the binding to and transactivation of Nanog-specific cis-regulatory sequences by Gli1 and 2.

In this regard, a number of additional regulatory mechanims of Hh/Gli function (e.g., Gli1 and 2 acetylation and HDAC ubiquitination and degradation by KCASH family of Cul3-dependent E3 ligases, Gli1 degradation by the Numb/Itch complex) play a role in the control of stemness, revealing a crucial role of Hh as a component of an integrated circuitry determining cell fate decision and involved in the maintenance of SCs.

Little is known about how cells coordinate their behavior to establish and regenerate functional tissue structure and restore micro-architecture. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing and 3D tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl₄, a prototypical inducer of pericentral liver damage and after hepatectomy. We have chosen the regenerating liver as an example because liver function depends on the complex micro-architecture formed by hepatocytes and microvessels, that is, sinusoids, which ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16-day regeneration process. The model unambiguously predicted a so far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process that we named ‘hepatocyte–sinusoid alignment’. The simulated tissue architecture was only in agreement with the experimentally obtained data when hepatocyte–sinusoid alignment was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the prediction, we analyzed the 3D orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. Meanwhile the technique of spatial-temporal modeling has been linked to metabolic and physiologically based pharmacokinetic models and the impact of certain types of tissue damage on metabolic performance have been correctly predicted. Furthermore, the model can be applied to predict repopulation of liver tissue after clinical transplantation of healthy hepatocytes into livers of patients with metabolic liver disease. We believe our procedure of spatial-temporal modeling is widely applicable in the systems biology of tissues.

There is no doubt that stem cell research will be one of the most promising approaches in medicine of the 21st century. It will probably revolutionize the therapy of many diseases including cardiac infarction and failure, diabetes, Parkinson’s disease and spinal cord lesion.

It is our aim to provide a fundamental basis for the development of new medical treatments. Stem cell research is a broad field that requires nearly all techniques of modern life science, including genetics, cell biology, physiology, biochemistry and histology but it also requires the input of experimental surgery and bioengineering technologies.

Induced pluripotent stem (iPS) cells represent the most promising approach for future stem cell-based tissue repair in regenerative medicine. iPS cells are functionally highly similar to embryonic stem cells, but in addition have the advantage of being ethically uncontroversial and obtainable from readily accessible autologous sources. However, although proof of principle for the therapeutic use of iPS cells in neuronal and cardiac diseases has been shown both at the laboratory scale and in animal models, the methods used today for generation, cultivation, differentiation and selection are not yet suitable for the clinic.

This presentation will give an overview of our recent research work on human embryonic stem cells in comparison with iPS cells. Starting from basic investigations on the physiological properties of cardiomyocytes developed from pluripotent stem cells we have established in vitro and in vivo transplantation models enabling us to systematically investigate and optimize the physiological integration and regeneration of the diseased tissue. Our main focus is the cardiac infarction model. Moreover, in vitro culture and expansion of stem cells is far from optimal and needs further research in order to overcome problems related to insufficient numbers of obtained stem cells and aging of the obtained stem cell population.

Regenerative medicine seeks to cure disease and injury by regenerating cells or tissues within the body. In general, therapeutic agents, whether small molecules, cells or complete tissues, work by rebuilding the diseased tissue or stimulating the body’s own reparative mechanisms to regenerate new tissue that ameliorates the underlying pathology. While both approaches can be very effective and often act by complementary mechanisms, it is imperative to understand the mechanism by which the therapy exerts its therapeutic effect in order to rationally design optimal strategies. We have pioneered bone marrow cell therapy as a treatment for osteogenesis imperfecta (OI), a genetic disorder of the bone that is typically caused by a mutation in one of the two genes that encode collagen I, the major structural protein in bone. Clinically, the severe form of OI is characterized by numerous painful fractures, marked bony deformities and short stature. While pharmaceuticals may alter the nature of bone to lessen symptoms, successful therapy for this genetic disorder can only be realized with cell or gene therapy focused on improving the quality of bone and the associated growth deficiency. To explore regenerative medicine approaches for bone disorders, we embarked on a program of translational research employing closely integrated laboratory and patient-based investigation. We first demonstrated that transplantation of unmanipulated bone marrow, effectively used to treat disorders of blood, could functionally engraft in recipient bone and provide measurable clinical benefits in children with severe OI. Based on the notion that marrow mesenchymal stromal cells (MSCs) generated the donor-derived osteopoiesis, we subsequently intravenously infused allogeneic, gene-marked MSCs obtained from marrow donors. Measured engraftment of the gene-marked MSCs in bone was associated with an acceleration of growth in the children; however, these cells also clearly demonstrated immune reactivity of MSCs in humans. In both clinical trials, mesenchymal engraftment was short-lived. Animal models of osteopoietic engraftment and differentiation suggest that nonadherent bone marrow cells, considered to be exclusively hematopoietic progenitors, may be superior to MSCs for systemic cell therapy of bone and the mechanism of osteopoietic engraftment may be an important factor governing the durability of the osteopoietic graft. This animal data led to our third clinical trial, which has unambiguously demonstrated that marrow mononuclear cell ‘boosts’ in OI children can lead to osteopoietic engraftment and striking clinical benefits in a subset of patients, suggesting potent mesenchymal progenitor activity within the hematopoietic compartment. Moreover, these data suggest that marrow mononuclear cells and MSCs benefit children with OI by differing mechanisms. In an animal model of OI, we demonstrate that the onadherent bone marrow cells engraft and differentiate to osteoblasts, contributing normal collagen to the OI bone matrix, while MSCs do not appreciable engraft; rather, these cells stimulate growth by the secretion of soluble mediators. Collectively, our animal and human data suggest that there are multiple mesenchymal progenitors and selecting the optimal cell may be an important factor in determining the success of cell therapy strategies. Currently, we are clinically investigating repeated infusions of MSCs in children with OI in an effort to maintain durable clinical benefits; while in the hematopoietic cell transplantation. The development of broadly applicable marrow cell therapy will depend, in part, on better knowledge of marrow cell biology, which will be most effectively uncovered by both innovative laboratory research as well as scientifically based clinical trials.

Objectives

Autologous bone marrow-derived mescenchymal stem cells (BM-MSCs) are now being used for treatment of equine tendon injury, but questions remain on the fate and function of the injected BM-MSCs. The ability to track the cells within the animal is important in understanding the regeneration process. The equine model, however, currently lacks ‘tools’ such as monoclonal antibodies to track, identify and analyze the fate of implanted MSCs.

Genetic labeling with green fluorescent protein is well established as a cell tracking marker and used in small animal experiments. However, transfection of green fluorescent protein in primary MSCs (not cell line), selection and expansion of the number of cells that emit good fluorescence is difficult and time consuming. The membrane dye CM-DiI (Molecular Probes, USA) has also been used frequently in cell tracking studies. CM-DiI contains a thiol-reactive chloromethyl moiety that allows the dye to covalently bind to cellular thiols. Thus, CM-DiI staining protocols are quick and easy and the label is well retained in some cells throughout fixation and routine paraffin processing.

Materials & methods

We performed CM-DiI labeling of equine BM-MSCs in vitro, noted its effect on cell division and studied dye retention in trilineage differentiation (chondrogenesis, adipogenesis and osteogenesis). In addition, we implanted CM-DiI-labeled BM-MSCs to the normal superficial digital flexor tendon (n = 2), naturally occurring lesion (n = 5) and surgically created lesion (n = 3) in the superficial digital flexor tendon. Tendons were recovered 3–170 days after implantation and the paraffin section or cryosection of tendon examined under a fluorescence microscope.

Results & conclusion

Viability of equine MSCs just after labeling by 1 µm MCM-DiI was 58.0 ± 16.6%. Fluorescence in cells disappeared after four passages. Labeled cells showed evidence of multipotency after labeling. Cells emitting fluorescence were identified in the damaged tendon for up to 170 days after implantation. When labeled cells were implanted, an extensive spread of cells was observed under ultrasound imaging. However, distribution within the tissue was limited, being mainly located in the endotenon with only small numbers present within the fascicles. There was no evidence of migration to adjacent areas of the tendon.

Most of the labeled cells retained phenotypic differences (more rounded morphology). However, some labeled cells on long-term tracing sections, over 130 days after implantation, had the same appearance as resident tenocytes with characteristic flattened nuclei. This suggested the implanted BM-MSCs could differentiate into tenocytes in vivo.

These studies demonstrated CM-DiI to be a simple and rapid alternative to genetic markers for tracking cells both in vitro and in vivo. We demonstrated that CM-DiI labeling provides a stable fluorescent tracking system and did not influence cell differentiation.

Objectives

The innovation concerns vascular implants of bacterial nanocellulose (BNC) [1] for heart bypass surgery and other blood vessels with an inner diameter of 6 mm or less. In size ranges like these, all traditional biomaterials fail mainly because of thrombosis. The biomaterial BNC is also of importance as a patch material for different surgical applications.

Materials & methods

Biotechnologically engineered as a form-stable hydrogel and built-up by a hierarchical 3D-nanofiber network of polymerized glucose (nanocellulose) and up to 99% water, the BNC implants represent a novel and exciting kind of biomaterial. Regarding this architecture, it is a polysaccharidic analog to the human body’s extracellular matrix collagen. The BNC biomaterial is highly poor, mechanically stable and easy to handle for surgeons, biocompatible, cannot be degraded by the human body, is vitalized after implantation and is nonthrombogenic. The properties and function-determining structure and shape of the tubular BNC implants are built by a patented matrix technology during biofabrication directly in the bioreactor and by avoiding shaping during postprocessing.

Results & conclusion

The result is a novel type of blood vessel implant with distinct bioactivity (in vivo scaffold), good transparency for liquids and ions, controllable water balance, variable diameter and length, vascular-like compliance and good suitability for sterilization and storage. The latest state of R&D efforts comprises prototypes, a specifically developed bioreactor and animal experiments as vascular grafts in rats (Dieter Schumann, Jena University, Germany) and sheep (Jens Wippermann, University of Cologne, Germany). The implants are endothelized at the inner surface and incorporated by the formation of connective tissue at the outer layer. Typical results and recent challenges will be presented and discussed. The successful use of BNC patches for closing ventricle septum defects are the result of a cooperative work with Nora Lang from Munich University, Germany (pig experiments).

In conclusion, BNC represents an innovative biomaterial with a great potential to solve open fundamental problems of medical implants, mainly in the field of blood vessel grafts. It combines, in an exciting manner, important features including its natural origin, high purity, high water content combined with good mechanical stability, good handling in all surgical steps and biocompatibility as well as bioactivity in the body.

A large number of diseases, from diabetes and heart failure to Parkinson’s disease, are caused by the loss of functional cells within the respective organ. Cell replacement therapy is now a widely discussed concept for the treatment of these types of pathologies. In many instances, terminally differentiated somatic cells are not well suited for transplantation; however, the use of stem cells, in particular pluripotent stem cells, is promising.

In my presentation, I will focus on concepts and recent progress in the treatment of Parkinson’s disease by transplantation of pluripotent stem cell-derived dopaminergic neurons. Parkinson’s disease is characterized by a loss of dopaminergic neurons of the substantia nigra. The dopamine-releasing nerve endings of these dopaminergic neurons are within the striatum. Thus, the pathophysiologically relevant consequence is a decrease of dopamine secretion within the striatum, leading to the typical movement disorder associated with Parkinson’s disease. Pluripotent stem cell-derived dopaminergic neurons have been shown to alleviate motor symptoms in Parkinson’s disease models in mice, rats and monkeys. In humans, Parkinson’s cell therapy has so far only been performed using fetal dopaminergic neurons. The outcome of the transplantation of Parkinson’s patients with fetal neurons was variable. From retrospective analyses, it appears that factors leading to good outcome included the use of freshly isolated cells, well dissociated cells (rather than tissue clamps) and prolonged immunosuppressive therapy. Yet, given the ethical and logistic problems associated with the use of fetal neurons, many experts believe that the use pluripotent stem cell-derived neurons might be a better solution.

While the rationale for developing a treatment for Parkinson’s disease with pluripotent stem cells is strong, an advancement towards clinical application still requires many issues to be solved. Key challenges include: robust generation of dopaminergic neurons under clinical-grade conditions, avoidance of death and/or dedifferentiation of the implanted dopaminergic neurons, avoidance of tumor formation (teratomas from undifferentiated pluripotent stem cells, or neural tumors from proliferating early neural precursor cells), optimized cell delivery and avoidance of immune rejection.

While it is likely that all these obstacles can be overcome, it is clear that concerted efforts are needed to bring the concept of Parkinson’s cell therapy into clinics. If done successfully, it would not only be an important progress in the treatment of a devastating neurodegenerative disease, but also a major breakthrough in the field of stem cell-based cell therapy.

Recent insights gained from studies of the developing cerebral cortex are illuminating potential evolutionary steps that contributed to structural and functional features of the human brain. Radial glial (RG) cells, long thought to simply guide embryonic nerve cells during migration, have now been identified as neuronal stem cells in the developing brain. RG cells undergo self-renewing, asymmetric divisions to generate neuronal precursors that can further proliferate in the subventricular zone (SVZ) to increase neuronal number. Unlike the developing rodent cortex, the developing human cortex contains a massively expanded SVZ (outer [O] SVZ) that is thought to account for the bulk of cortical neurogenesis. We have begun to characterize the types and locations of progenitor cells responsible for human cortical development. We found that large numbers of RG-like cells and intermediate progenitor cells populate the human OSVZ. The OSVZ RG-like cells have a long basal process but, surprisingly, do not have basolateral polarity and lack contact with the ventricular surface. Using real-time imaging and clonal analysis, we demonstrate that these cells undergo self-renewing, asymmetric divisions to generate neuronal progenitor cells that can further proliferate. We have recently found that progenitor cells resembling outer RG cells are present in mouse embryonic neocortex and arise from asymmetric divisions of the RG. Time-lapse imaging reveals that the cells undergo self-renewing, asymmetric divisions to generate neurons. These results suggest that oRG cells are probably present in all mammals and are not a specialization of a larger brain with increased cortical area. Instead, an evolutionary increase in the number of oRG cells and their transit amplifying daughter cells likely amplified neuronal production and contributed to increased cortical size and complexity in the human brain. Moreover, this pattern of neurogenesis suggests strategies for generating large numbers of specific neurons for cell-based therapies.

The development of biomaterials follows the demands of clinical applications. In regenerative medicine biomaterials are essential, especially when large defects have to be restored [1,2]. Biomaterials established in clinical application nowadays cannot completely fulfil the complex requirements of regenerative therapies, as each application requires a specific combination of intrinsic properties (e.g., elasticity) and functions (e.g., degradability or biofunctionality). Another challenge is the predictability of the long-term behavior of biomaterials in biological environments [3]. Biomaterial-based regenerative therapies include degradable implants inducing endogenous regeneration and scaffolds as temporary extracellular matrix substitutes for tissue engineering applications. Implants such as surgical sutures or hernia meshes had initially been developed for a specific mechanical/structural performance. With increasing clinical experience it became apparent that one single function is not sufficient, but multifunctionality is required [2]. Vascular stents, which were purely metallic devices with a specific structural function in the beginning, have been further developed by adding polymeric coatings to improve their hemocompatibility. This coating was partially loaded with drugs to avoid restenosis. Presently, degradable stents are under development. In this presentation, the scientific challenges of combining several functions in one material system are described and examples for dual- and triple-functional polymer systems are given [4–6]. Finally, the application potential of multifunctional shape-memory polymers in regenerative medicine is outlined [4,7,8].

Despite the compelling clinical need to regenerate damaged tissues/organs, impressive advances in the field of tissue engineering have yet to result in viable engineered tissue products with widespread therapeutic adoption. The main challenges to be overcome have been identified in the as yet not convincing benefit of the proposed therapies, combined with their high costs. Following the exemplifying paradigm of bone and cartilage regeneration, the lecture will highlight the bottlenecks of typical manufacturing strategies and will propose alternative bioreactor-based approaches for the manufacturing of 3D cellular grafts. The perspective will address issues related to quality standardization, process control and regulatory compliance in manufacturing cell-based products and highlight the need not only to automate, but also to streamline and simplify typical production processes. Examples will be given on the attractive paradigm to expand mesenchymal stem/progenitor cells from adult individuals directly in a ‘3D niche’ environment, thereby maintaining a larger postexpansion differentiation capacity and bypassing the complex and costly serial cell passaging in monolayers. Finally, as a next-generation paradigm, the lecture will propose and exemplify the concept of engineering regenerative strategies following principles of developmental biology, using the body as the in vivo bioreactor.

We have recently developed a reprogramming method utilizing transposon-mediated delivery of the reprogramming transgenes. This system has several advantages over the viral delivery-based alternative. Most notably, it allows for a seamless removal of the transgenes once induced pluripotent stem (iPS) cells have been generated and they are no longer needed for stem cell self-renewal. We also combined the doxycycline-inducible transgene expression system with the transposon delivery-based reprogramming and found that these transgenes are very efficiently regulatable by adding or withdrawing doxycycline. In vivo differentiated somatic cells derived from iPS cells can be reprogrammed to ‘secondary’ iPS cells by simply adding doxycycline to the culture medium. Somatic cell lines produced with this method frequently return to secondary iPS cells in a population manner, which allows us to study the cascade of molecular events during the entire process of reprogramming with proteomics, genomics and epigenomics.

Because adult lung tissue has limited regenerative capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue and when implanted into rats in vivo for short time intervals (45–120 min), the engineered lungs participated in gas exchange. Although representing only an initial step towards the ultimate goal of generating fully functional lungs in vitro, these results suggest that further investigation of this approach is warranted.

This presentation focuses upon the market analyses Select Biosciences has been conducting to characterize the various segments of the worldwide stem cells market. Data presented in this talk describe historical market trends – both qualitative and quantitative – as well as forward-looking projections for the stem cells’ space based upon bottom-up analysis of the various individual spaces that taken together compose the current worldwide stem cells marketplace.

The segments that will be presented are regarding pluripotent stem cells, cord blood stem cells and the various adult stem cell types. Market opportunities for each of these stem cell types have been analyzed in Select Biosciences syndicated market analyses, and in this talk, vignettes from these analyses will be presented and framed in the context of the broader cellular therapy space and the evolution of this field.

Achieving tolerance towards foreign grafts and thus preventing permanent immunosuppression with all the known severe side effects is the most important goal in transplantation medicine. In the last 20 years, major progress has been made in understanding the mechanisms that regulate tolerance induction towards foreign grafts in small animal models. However, rarely can such knowledge be translated into the development of successful new therapeutic approaches in clinical transplantation. The success is limited by clinical challenges that are not present in our clean animal facilities such as heterologous immunity – and the pathogen-specific memory T and B cells recognize alloantigens and boost the immune response towards the allograft presensitization of recipients – presence of allospecific memory T and B cells that are inert to most known therapeutic regimens. Thus, we know now that we need more personalized treatment strategies according to the patient’s immune reactivity. Such a strategy should combine three important aspects: improved immune monitoring; treatment that targets memory cells; and strategies to reinforce regulation.

We established preclinical transplant models with preformed alloreactive or pathogen-specific memory T cells in which we compare the effectiveness of different treatment approaches combining depletional with regulatory approaches.

Furthermore, we have performed a DNA microarray screen on samples of transplant patients and identified surface molecules specifically expressed by naive, central memory, effector memory or terminally differentiated effector memory T cells. Using this approach, we hope to develop antibodies, that specifically deplete effector and terminal differentiated effector memory cells but spare naive and central memory T cells and thus be associated with less side effects compared with a global depletion of T and B cells.

With the publication of the microarray quality control-I and -II studies in 2006 resp. 2010, transcriptomics made a big step forward towards truly diagnostic use. The reliability of the technology itself and of the mathematical tools developed so far make this approach a prime candidate to improve our diagnostic capabilities in the near future. However, the lengthy process of developing molecular signatures for diagnostics or even prediction make this endeavour cumbersome. To address these issues, we have developed combined simulation and adaptive learning approaches for a faster development of molecular signatures for diagnostic use. With only limited data available from pilot trials, we now can simulate results in up to 10,000 clinical trials. Using adaptive learning approaches we can estimate the number of samples needed for larger validation trials and thereby can speed up the approval process.

Applying these new approaches, we have successfully developed a diagnostic test for primary diagnosis of leukemias and can now estimate the performance of future diagnostic tests at recognizing patients with active tuberculosis, HIV infection or early lung cancer in peripheral blood.

Objectives

Healing after tendinopathy is associated with the formation of scar tissue within the tendon, characterized by increased structural stiffness, a disorganized matrix and accumulated glycosaminoglycans, and is associated with functional deficits. Mesenchymal stem cells (MSCs) offer the potential for improved functional outcome and are now widely used to treat naturally occurring overuse tendon injury in horses. The objectives of this study were to determine cell survival after implantation and to evaluate efficacy at both tissue and whole-animal levels.

Methods

Survival after implantation

Mesenchymal stem cell-labeled with technetium-99m pertechnetate linked to hexamethylpropyleneamine oxime (CeretecÔ) were followed for 36 h after intralesional, intravenous and regional perfusion injection.

Effects on tendon matrix

Two groups of five horses with naturally occurring superficial digital flexor tendon (SDFT) injury received either 1 × 10⁷ autologous MSCs in 2 ml bone marrow supernatant or saline (control). Horses received controlled exercise and were euthanized after 6 months. Nondestructive mechanical testing and morphological and compositional analyses were performed on the SDFT.

Clinical outcome

Autologous MSCs were implanted into 141 racehorses with naturally occurring SDFT injury. Reinjury rate over a 3-year period after treatment was compared with two published studies [1,2].

Results

Survival after implantation

Mesenchymal stem cells were retained focally after intralesional implantation but only approximately 15% of cells remained 24 h after implantation. Regional perfusion exhibited more limited labeling in 11 out of 12 tendon lesions but no labeling was seen after intravenous administration.

Effects on tendon matrix

Mechanical testing, organization and molecular analysis of the new tissue showed ‘normalization’ of the healing tissue towards that of normal tendon compared with controls.

Clinical outcome

Reinjury rates for MSC-treated national hunt horses was significantly lower (25.7%) than in conventionally managed horses (56 and 53%; p < 0.05).

Conclusion

There is a rapid loss of cells after implantation but some cells do remain within the injured tissue, which appear to induce a normalization of biomechanical, histological and compositional parameters towards those in normal (or less injured) tendons. This is supported by a reduction in reinjury rate in an adequately powered clinical study (although without contemporaneous controls). Thus, this technique appears to be safe and supports the potential use of MSC’s in human tendon injuries, which share many similarities with equine tendinopathy.

Funding

Horserace Betting Levy Board, The Royal Veterinary College, Consejeria de Innovacion Ciencia y Empresa, Junta de Andalucía, Spain, and Quy Biosciences Ltd (VetCell).

Disclosure

R Smith is a technical adviser for VetCell.

Thanks to progress in genomics, proteomics and biomedicine, it is becoming clear that diseases, as well as their potential cures, are associated with specific processes at cellular and molecular levels. The scientific challenge is to achieve an accurate understanding of disease pathways at these levels, which is the focus of an emerging area of research known as systems biology.

Molecular imaging is a fast-developing field utilizing these insights and provides tools and applications for the early detection of pathological processes associated with a given disease at the cellular and molecular level rather than at anatomical level as in ‘classical’ diagnostic imaging. In the foreseeable future, clinical applications of in vivo molecular imaging as well as in vitro molecular diagnostics – two building blocks of molecular medicine – will be used for earlier detection and better staging of diseases, more accurate therapy planning and monitoring, and improved follow-up care.

Key to molecular imaging is the development of targeted (contrast) agents that bind selectively to specific molecules or at specific sites in the body. The development of new contrast agents and, ultimately, pharmaceutical remedies that can be specifically directed to those places in the body where there is evidence of the disease enables doctors to treat patients more effectively and to observe the results of the treatment in the individual patient. These agents are visualized with the help of imaging systems such as PET, MRI, optical bioluminescence and fluorescence, ultrasound and others.

Similarly, the shift in recent drug discovery to novel compounds against specific molecular targets highlights the need for techniques to visualize and follow drug–target interactions and biological processes in vivo on a molecular level. Therefore, the potential for molecular imaging in the preclinical stage is significant. As a noninvasive technique, it can be repeated many times to provide both spatial and temporal dimensions to the understanding of disease or therapy in animal models.

Finally, we explain the need for precompetitive collaborations and networks to advance the development of biomarkers, biosignatures and appropriate detection technologies and assays.

Cardiac stem cell therapy aims at the repair of impaired myocardium to prevent ventricular remodeling and to improve overall physical performance. Since the first-in-man use of bone marrow stem cells after myocardial infarction in 2001, a large number of clinical studies have demonstrated their safety and clinical efficacy. Cardiac stem cell therapy can be performed intracoronary or intravenous with cardiac catheterization techniques, as well as also easily during cardiac surgery interventions. The degree of severity of heart failure patients, as well as their physical activity improved over all other therapeutic regimens. Cardiac stem cell therapy represents an ultimate approach in advanced cardiac failure. In acute myocardial infarction and chronic ischemia, long-term mortality after 1 and 5 years, respectively, is significantly reduced. A few studies also indicate beneficial effects in chronic dilated cardiomyopathy. With the use of primary bone marrow stem cells, there are no major stem cell-related side effects, in particular, no cardiac arrhythmias and inflammation. New cell isolation techniques allow point-of-care cell preparations within the cardiac intervention or operation theater, thereby providing short preparation times, facilitated logistics of cell transport and reasonable cost–effectiveness of the whole procedure. The main indications for clinical treatment are acute infarction, chronic ischemic heart failure and dilated cardiomyopathy. Further multicenter and randomized trials are needed to elucidate the mechanisms of stem cell action and to extend the current use of intracoronary and/or intramyocardial stem cell therapy.

Objectives

Soluble CD83 (sCD83) is a novel immunomodulatory molecule that has been shown to interfere with dendritic cell-mediated T-cell proliferation in vitro. In order to investigate the in vivo efficacy of sCD83, the murine experimental allergic encephalomyelitis (EAE) model, as well as skin and heart transplant models, were used.

Materials & methods

For the induction of EAE, mice were immunized with myelin oligodendrocyte glycoprotein peptide in combination with CFA and pertussis toxin on day 0. sCD83 (100 µg/mouse i.p.) was either applied in a prophylactic or in a therapeutic setting. In the minor mismatch skin transplantation model, male donor skin was transplanted onto the back of female recipient animals. Recipients were either treated with sCD83 (day 1 until day 7) or were left untreated. In addition, the immunomodulatory effect of sCD83 was investigated using an allogeneic murine heart transplant model in combination with anti-CD45RB mAb and rapamycin. Thus, C3H mouse hearts were transplanted into C57BL/6 mice.

Results & conclusion

In the murine EAE-model, we demonstrated that sCD83 is able to inhibit the paralyses in a prophylactic as well as in a therapeutic setting. Using the murine skin transplant model, we demonstrated that sCD83 prevented the transplant rejection in 50% of the treated animals. Next, the sCD83-treated animals that did not reject the first transplant were transplanted for a second time and, strikingly, all transplants were accepted even though sCD83 was not applied during this second transplantation, indicating that sCD83 induces regulatory mechanisms, possibly Tregs. Encouraging results were also obtained in the heart transplant model: without immunosuppression, heart grafts were rejected in 8.5 days. sCD83 monotherapy attenuated acute rejection and doubled heart graft survival to 15.1 days. In addition, sCD83 has synergy with subtherapeutic doses of either anti-CD45RB mAb or rapamycin to further improve graft survival to 32.0 and 39.2 days, respectively. Remarkably, sCD83 in combination with both anti-CD45RB mAb and rapamycin effectively prevented acute rejection and achieved graft tolerance with indefinite survival. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days. Success of triple-therapy treatment was accompanied by enhancement of tolerogenic dendritic cells that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. Thus, this study revealed that sCD83 is capable of inducing donor-specific allograft tolerance without toxicity.

Salamanders regenerate an entire limb after amputation and investigating how injury initiates a coordinated regeneration of bone, muscle, nerve and skin can provide important insights into tissue engineering and regenerative medicine. Here, we show that connective tissue cells and muscle cells have differing behaviors during limb regeneration. Connective tissue cells provide a strong patterning influence that shapes the pattern of the regenerating limb, whereas muscle plays a more promiscous role in patterning. Furthermore, the upregulation of important transcription factors for regenerating a patterned limb in the two systems show important differences. Finally, we have investigated the role of signals from the nerve in the induction of regenerative factors.

The function of musculoskeletal tissues is largely, if not entirely, biomechanical. Most orthopedic lesions are the consequence of a mismatch between biomechanical loading and the resistance to these loads of the tissues involved. For this reason, if regenerative medicine is applied to musculoskeletal tissues, the biomechanical qualities of the artificial tissues that are created in this regeneration process are paramount.

Until recently, the emphasis in the design of scaffolds that are to be used to substitute tissues and/or are meant to form the starting point for enhanced autologous repair has been on their biological qualities rather than on biomechanical characteristics. This has been a severely limiting factor in the eventual clinical application of scaffold technology in regenerative medicine of musculoskeletal tissues.

The advent of printing techniques and the development of hybrid scaffolds composed of components with widely varying biological and biomechanical features have opened new ways to overcome this problem.

Providing printed hybrid constructs that are based on the use of materials with different biomechanical properties and are characterized by a zonal organization may improve long-term outcomes of attempts at cartilage repair via tissue engineering techniques, since chondrocyte–matrix interactions are essential for the maintenance of the zonal chondrocyte phenotype. Moreover, it has been demonstrated that isolated and expanded chondrocytes from the different depth zones of the cartilage regain their zonal differences when they are redifferentiated in alginate beads, as evidenced by zone-specific reappearance of cartilage oligomeric matrix protein and clusterin, as well as significantly higher GAG production by cells from the deep compared with the superficial zone.

To achieve this, we are employing organ-printing technology or bioprinting, which combines the deposition of specific cell populations with the simultaneous deposition of biomaterials. This allows the development of zonal cartilaginous grafts and, by using hydrogels combined with (much stiffer) polymers, a more physiological environment can be created. Adding biologically active components, such as proteins, peptides, DNA, hormones, extracellular matrix molecules and natural or synthetic polymers, to these water-based bio-inks will further enhance and direct the behavior of the cells. We characterized the use of bioprinting technologies to design and build heterogeneous cell-laden 3D structures and evaluated these in vitro and in vivo.

Objectives

To evaluate mesenchymal stem cell (MSC) distribution and persistence after intralesional (IL) injection, intra-arterial (IA) and intravenous (IV) regional distal limb perfusions (RLPs) using scintigraphy in normal horses and after surgical induction of lesions in superficial digital flexor tendons.

Materials & methods

Six anesthetized control horses were used to assess IV and IA RLP of technetium (Tc)-hexamethylpropyleneamine oxime (HMPAO)-labeled MSCs in the equine distal forelimb through the median artery of one limb and the cephalic vein of the contralateral limb, at the level of the distal radius. A pneumatic tourniquet was applied proximaly to the catheter for 45 min, just prior to injection. Scintigraphic images were acquired at the time of injection and after 40, 45, 75 min and 6 and 24 h. MSC persistence (%) was calculated at each time point as the ratio of the activity in the limb after correction for decay and activity in the limb immediately after injection. Tendon lesions were then surgically induced in the superficial digital flexor tendon at 13 and 20 cm below the accessory carpal bone in both forelimbs of eight horses. In six horses, Tc-HMPAO-labeled MSCs were administered 3 days after surgery using IL injection, IV RLP or IA RLP (four limbs for each treatment). In two horses, IA and IV RLP was performed 10 days after surgery.

Results

The IA RLP resulted in diffuse distal limb distribution of labeled MSCs in all control horses, but IV RLP led to poor distribution or absence of MSCs in the pastern and foot of three horses. Initial cell persistence was 100% for both RLP techniques, but decreased to as low as 28% after 6 h. In the 3-day-old lesions, IL treatment showed a larger number of MSCs at the lesion site when compared with the RLP treatments, but the perecentage persistence was similar to IA and IV RLP. Persistence in limbs with lesions was similar to that in normal limbs for both IA and IV RLP and similar within lesions compared with the entire limb. In 10-day-old lesions, however, persistence within lesions was higher than in the rest of the limb and higher than in 3-day-old lesions for the first three time points (40, 45 and 75 min). After 6 and 24 h, persistence at the lesion site was similar to the rest of the limb. The overall persistence within the limb with the 10-day-old lesions was similar to that of the limbs with 3-day-old lesions. Finally, IA PRP has consistently led to thrombosis in the metacarpal arteries and, unlike IV RLP, occasionally led to clinical complications.

Conclusion

Scintigraphy of Tc-HMPAO-labeled MSCs showed that both IA and IV RLP led to persistence of stem cells in the distal limb. However, while IA RLP distributes MSCs more reliably and diffusely via in the distal limb, it has been associated with clinical complications. No significant cell homing within 3-day-old lesions was detectable, but the increased persistence of MSCs in 10-day-old lesions suggests a potential time-dependent homing effect on stem cells.

Biomarker discovery and development is an important and steadily growing field in modern life sciences. Assays based on these biomarkers have become more and more important with respect to correct diagnosis and personalized decisions in the therapy of complex diseases such as cancer. Only optimal preserved sample material allows successful biomarker discovery. Sample handling, storage and processing are often neglected processes, even though their influence on assay results can be critical. For example, artificial modifications of the RNA content and profile in blood samples post-phlebotomy caused by degradation and gene induction is well documented. Similar artefacts can be expected if unstabilized or formalin-fixed tissue samples are used for biomarker discovery. Doubtful or inconsistent results are the consequence, especially for quantitative or semiquantitative analytical methods such as quantitative real time-PCR assays and microarrays. In my talk, I will focus on technical prerequisites, hurdles and solutions for optimal sample processing.

Question

Bioreactor design in tissue engineering is complex and at the early stages of its development. Design of biologically effective, yet scalable, devices requires the intimate collaboration between engineers and biologists. Growth conditions, harvesting time, scale-up, storage and sterility issues all need to be considered and incorporated into design of bioreactors and machines for the automatization the production of human tissues.

Methods

Bioreactors can be designed to maintain physiological parameters at desired levels, enhance mass transport rates and expose cultured tissues to specific stimuli. The requirements of functional tissue engineering include:

• ▪ Cellular components capable of differentiation into appropriate lineages;

• ▪ A scaffold providing a structural template for tissue development;

• ▪ A bioreactor providing the necessary biochemical and physiological regulatory signals guiding cell differentiation and tissue development.

To ensure homogeneous conditions within the complete area of bioreactors, methods of topology and shape optimization are applied. The results are compared with analytical models, from which a general parametric description of the design is obtained and tested.

Results

During the talk, a state-of-the-art review of specific reactors and the automatization of single production steps and whole processes will be provided. A major topic will be the discussion of concepts that are important in the development of reactors and technologies that can be used for the production of clinical scale tissue. At the end of the talk, a general overview of a fully automated process to produce skin equivalents will be given.

Conclusion

Without a comprehensive understanding of each of these components, bioreactor design and tissue growth to manufacture product will remain at a relatively rudimentary and limited level. Increased fundamental understanding of the issues can have a dramatic impact on the ability to generate tissue-engineered product safely, economically and in the numbers that are required to fully address the patient populations in need.

DNA methylation of polycomb group target genes (PCGTs), which are required for differentiation of human embryonic and adult stem cells, is one of the hallmarks of human cancer. Evidence so far supports a model of carcinogenesis in which stem and progenitor cells gradually acquire de novo methylation at PCGTs, which eventually locks these cells in an undifferentiated state of self-renewal and consequently predisposes them to malignant transformation. Aging is a major modulator of methylation at PCGTs. We have found that PCGTs are heavily enriched among these CpGs that become hypermethylated with age and that methylation of these ‘age PCGTs’ is one of the first molecular events occurring during human carcinogenesis. Recently, it was shown that long noncoding RNA HOX antisense intergenic RNA (HOTAIR) recruits polycomb-repressive complex 2 to specific PCGTs. In ovarian cancer, we have shown that DNA methylation at PCGTs is associated with substantially reduced chemosensitivity, which in turn leads to poor survival in patients and, fundamentally, that this effect is specifically observed in tumors overexpressing HOTAIR.

Polycomb-triggered gene regulation occurs predominantly at the level of histone methylation. DNA methylation is another epigenetic phenomenon that occurs in human embryonic stem cells. A substantial fraction of CpGs are (>80%) methylated human embryonic stem cells (MESCs). These MESCs become specifically hypomethylated during carcinogenesis, in particular during the transition from intraepithelial neoplasia to invasion, suggesting that methylation at MESC CpGs is an essential mechanism by which stem cells prevent invasion.

In summary, epigenetic regulation of MESC and PCGT genes is crucial for stem cell homeostasis, and epigenetic disturbance of this process plays a major role in neoplastic transformation.