Final Report Summary - STELLAR (Stem-cell based therapy for kidney repair) Executive Summary:The central hypothesis of the STELLAR project is that kidney stromal cells (kPSCs) confer specific regenerative and homeostatic functions with respect to kidney regeneration as compared to mesenchymal stromal cells (MSC) isolated from other tissues such as bone marrow or umbilical cord. STELLAR has delivered a clinical grade GMP compliant protocol for the isolation, sorting and culture expansion of kPSC from human kidneys not fit for transplantation that would otherwise be discarded. kPSC isolated from these kidneys were compared to MSC isolated from other tissues, bone marrow and umbilical cord, with respect to phenotypical and functional differences. Although MSC derived from different tissues look phenotypically alike, functional differences were apparent. Most importantly, kPSC have a potent renoprotective activity, more so than ucMSC and bmMSC, on the tubular injury in an animal model that closely resembles chronic kidney disease development. kPSC infusion decreased the percentage of glomeruli affected by fibrotic lesions and glomerulosclerosis. Factors possibly responsible for fibrosis, which were found to be markedly increased in this animal model of kidney disease were normalized by the infusion of kPSC-derived conditioned medium, indicating an anti-fibrotic activity of the renal stromal cell therapy. Taken together results obtained in STELLAR show that kPSC cell therapy or kPSC conditioned medium therapy is more effective in a representative model of chronic kidney disease compared to bmMSC or ucMSC therapy. The mechanisms of action responsible for the protective effect of MSC/kPSC in kidney disease were unraveled. The STELLAR consortium received funding in a call geared towards promoting the collaboration between European and Australian institutes. The collaborative efforts between the two continents went beyond the work performed within STELLAR and new collaborative actions spinning off from the STELLAR work have been initiated and continue to exist after the closure of the STELLAR project. With so many patients suffering from end stage renal disease, disseminating the STELLAR project to patients and lay people using Facebook has been quite rewarding. We have built up a solid group of followers and will keep providing these patients with the latest developments within the kidney regenerative medicine field also after the project closure. The STELLAR consortium partners look back on a successful project period and hope to find the means to collaborate for the better of kidney disease patients in the future. Project Context and Objectives:Chronic kidney disease (CKD) affects 8% of the European population and ultimately results in renal failure due to progressive fibrosis. CDK carries a high mortality risk and the number of affected people rises, increasing the demand on renal replacement therapies while the number of available donor organs stays stable.The STELLAR consortium proposes to develop an alternative to renal replacement therapy, based on the repair capacity of newly discovered kidney mesenchymal stromal cells (kPSCs). By injecting kPSC into affected kidneys, we expect to stop kidney fibrosis and induce tissue repair, ultimately leading to the restoration of normal kidney function. The STELLAR consortium will:• develop protocols for up scalable, high quality isolation of kPSCs and precisely characterize kPSC function in comparison to other MSCs.• test kPSCs in several murine renal disease models, to study their effects on fibrosis and tissue repair.• discover mechanisms of kidney repair.• invest in developing the technology necessary for up scaled isolation and quality control.The STELLAR consortium combines Australian experts on kPSC isolation and characterisation with European experts on renal failure and compounds the state-of-the-art knowledge, facilities and experience needed to develop and validate this novel form of renal therapy. The inclusion of experienced SMEs, with great technical and scientific know-how about assay and protocol development, further strengthens the consortium and will ensure not only the inclusion of new technology, but also a quick translation from bench to clinical application.Project Results:Results obtained during the five years of the STELLAR project with respect to the above formulated objectives will be discussed per work package. Almost all objectives and milestones set forward at the start of the project have been achieved and all deliverables have been submitted to the commission.WP1: To isolate and characterise of endogenous kPSC from human tissue and their functional comparison. In this WP the optimal strategy to isolate human MSC from the kidney, termed kPSC, was analysed. These cells were characterised and compared to MSC isolated from bone marrow and umbilical cord. An isolation procedure to isolate crude cells from the kidney was designed which encompasses perfusion of a discarded kidney with digestive enzymes to obtain a single cell suspension via the renal artery. This cell suspension is than further enriched for kPSC using an NG2 directed antibody for positive cell sorting. Sorted cells can be culture expanded either in tissue culture flasks and/or using a closed bioreactor system. All individual steps in this process: crude cell isolation, NG2 sorting and bioreactor culture expansion, were optimised and translated to a GMP compliant isolation procedure in WP3.With respect to cell surface marker expression the consortium showed that all three tested cell types showed a similar marker profile being positive for typical MSC markers CD73, CD90, CD105, NG2 and negative for CD34, CD45, CD31, CD56. Differences between kPSC and bone marrow derived MSC were observed with respect to differentiation capacity. while bmMSC can differentiate into bone, fat and cartilage, kPSC do have osteogenic and chondrogenic differentiation potential but do not differentiate into adipocytes. As MSC are mainly delivered by intravenous infusions, efficient homing of these cells to the site of injury might be critical to exert their anti-inflammatory and immunosuppressive/regenerative properties. With respect to homing and migration kPSC and ucMSC showed a more efficient motility in response to TNF-α as compared to activated bmMSC thus suggesting their greater intrinsic ability to migrate. Furthermore, kMSC and uc-MSC when injected in vivo as cell therapy may represent a more efficient cell population in respect to bmMSC, based on their capacity to adhere to dysfunctional endothelium and to transmigrate toward damaged tissues thus supporting a stronger regenerative process. More interestingly, using an epithelial wound healing model, the paracrine effects of kPSC and ucMSC were shown to be much higher compared to bmMSC. The immunemodulatory potential of all three cell types was shown to be comparable. The mRNA and miRNA profiles of the three cell types show distinct differences and hierarchical clustering was observed per cell type.One of the tasks in WP1 was to design a topography or 3D culture substrates that can maintain or induce the desired MSC phenotype. So far, this objective is not achieved. Results obtained showed that MSC are best cultured on a 2-D flat surface in which they display the highest proliferation and secretion of cytokines of interest. A feasibility study to transfer topography to micro carriers used in bioreactors showed that the currently used techniques are not transferrable. Furthermore, carriers currently used for cell expansion in the bioreactor are degradable beads not suitable for 3D topography transfer. WP2: To discover the role and mechanism of action of kPSC in normal kidney homeostasis and kidney repair. In this WP the in vivo role of endogenous kPSC in kidney physiology and response to injury was investigated. Furthermore, kPSC isolated from mice and the human counterpart were compared for their functional equivalence. In depth mechanistic studies in mice showed a hitherto unknown plasticity of kPSC within the adult kidney. kPSC in mice were shown to actually arise from the collecting duct epithelium, and at least a portion of cells (<1%) from the adult collecting duct can undergo EMT to grow as colony forming clonogenic stem cells with MSC properties, but can also revert specifically to collecting duct when reintroduced to the neonatal kidney. In vitro epithelial repair was shown for kPSC in an epithelial wound healing assay. Murine kPSC like cells expressing HoxB7 were shown to have a higher epithelial repair capacity compared to their non-HOXB7 expressing counterparts. The superior epithelial wound healing capacity was also shown for human kPSC. The mechanism underlying this capacity can in part be contributed to the expression of hepatocyte growth factor as HGF blocking antibodies reduced this effect. In vivo repair capacity of kPSC compared to other MSC types differs in the various mouse models used in WP3. Furthermore, mouse kPSC are phenotypically similar to mouse bmMSC and have similar differentiation capacities but differences in mRNA expression profiles were observed. Differences in in vivo behaviour were observed. When injected into neonatal mice kPSCs were able to integrate within the collecting ducts of the kidney while bmMSCs were not. Differences between mouse and human kPSC were observed in the site of integration in vivo. While mouse kPSC integrate into collecting ducts, human kPSC were observed to integrate more proximal, most likely in the proximal tubuli.WP3: The in vivo therapeutic potential of kPSC in representative models of progressive kidney disease.In this work package MSC derived from different tissues were tested and compared for their effects in models of kidney disease. Putative mechanisms of action were studied and a biomarker profile representative of renal repair was explored. The animal models used showed different results with respect to which cell type could be best used to treat kidney disease. In an ADR-induced nephropathy rat model bm-MSC, ucMSC and kPSC were shown to have an anti-inflammatory effect, inducing polarization of macrophages from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype. The effect was most prominent when ucMSC were used. Using a renal ischemia reperfusion model no decrease in kidney injury was observed between control animals and animals that received either bmMSCs or kPSCs, which was shown using both blood urea nitrogen levels and staining for the tubular injury marker KIM-1. The model that best reflects chronic kidney disease, is a renal mass reduction (RMR) model in rats. This is an established CKD model characterized by glomerular hypertension, enhanced protein ultrafiltration, accompanied by glomerular structural changes. Data obtained shows that all the treatments significantly reduced glomerular podocyte loss and endothelial injury in response to RMR. Importantly, kPSC therapy was more effective in limiting the progression of proteinuria over time as well as the formation of glomerular synechiae. Consistent with the inhibition of the intercellular podocyte-PEC bridges, kPSC infusion also decreased the percentage of glomeruli affected by fibrotic lesions and glomerulosclerosis. Factors possibly responsible for fibrosis, were found to be markedly increased in sera of RMR-rats which was normalized by the infusion of kPSC-derived conditioned indicating an anti-fibrotic activity of the renal stromal cell therapy. RMR also affected non-glomerular structures including tubular casts, dilatation and atrophy, which were reduced by kPSC treatment, though not in a significant manner. In support to these data, the observation that urinary KIM-1, a biomarker of tubular injury, increased in RMR rats and was significantly reduced by kPSCs and the corresponding conditioned medium, highlights that these renal precursors have a potent renoprotective activity, more so than ucMSC and bmMSC, on the tubular injury in this model. We can conclude that kPSC cell therapy or kPSC conditioned medium therapy is more effective in a model of chronic kidney disease compared to bmMSC or ucMSC therapy. The observation of the reduction of pro-inflammatory biomarkers in urine samples upon stromal cell therapy treatment is valuable with regard to gaining a better understanding of the progression of kidney diseaseWith respect to the mechanisms of action some very exciting and controversial results were obtained. These results are still confidential and currently not publically available. WP4 To develop a first clinical protocol for kPSC based cell therapy for progressive kidney diseaseThe goal of this work package was to finalise the development of a bioreactor for GMP cell production at a clinically relevant scale. A new carrier type was introduced which greatly improved the expansion capability of MSC. This opened the path to further develop a closed system bioreactor to the state where it can be placed on the market in 2018 after validation runs have been completed. In the Scinus bioreactor, expansion of MSC has been performed keeping in mind the requirements for clinical production. All steps minimize operator involvement and every operation is “closed” where possible. All additions to the Scinus Cell Expansion system (MSC-enriched cell population, medium, microcarriers, etc.) can be done by sterile welding, thereby maintaining a closed environment. Medium refreshment and harvest of the entire cell population is similarly performed in a closed manner and operator involvement is limited to switching containers. As a result, operator time could be reduced more than 75% compared to monolayer expansion.The NG2 separation technique set up in WP2 was translated into the CliniMACS Prodigy, a closed GMP compliant and automated cell processing device. Starting from a clinical grade crude cell extract isolated from discarded kidneys, kPSC showed comparable morphology and phenotype as previously revealed in small scale experiments. It was successfully shown that the serum- and xeno-free GMP compliant MSC-Brew GMP medium can be used for the propagation of kPSCs with stable expansion rates and phenotype. A subsequent upstream processing and large scale expansion of kPSC with the Scinus Bioreactor is easily feasible. WP5 Exchanging knowledge.This work package addressed the exchange of knowledge between consortium members from two different continents, EU and Australia. The STELLAR project and the Australian-European collaboration has created a number of unique scientific breakthroughs and really fascinating economic opportunities. Besides the collaborative work performed on the STELLAR grant a number of other collaborative projects were initiated over year between the partners. WP6 Dissemination and exploitationThis work package served to inform all stakeholders of the progress and findings within the STELLAR consortium. A website was generated and a Facebook page was set up that accumulated 480 followers over the years. Various television broadcasts, a movie on the isolation of kPSC and the organisation of the STELLAR end symposium are examples of dissemination efforts. We are very proud of a song specifically composed for STELLAR by a kidney disease patient. STELLAR produced quite some scientific output and we expect more scientific papers to appear within the coming 6 months.WP7 Management and coordinationManagement of the project was kept as lean as possible with consortium meeting every 9-12 months. Telco’s were organised when necessary. The project ran quite smoothly, and management was focussed on promoting interactions between partners and follow-up of the work. Some modifications to tasks and budgets were discussed with the general assembly. Deliverables were provided to the coordinator for uploaded well in time before the delivery deadlines. A number of amendments were requested mainly due to relocation of partners. The project has achieved most of its project goals and no significant deviations from the overall plan were necessary.Potential Impact:The STELLAR consortium strived towards a better understanding of the mechanisms of kidney repair using MSC therapy. Understanding the basic mechanisms of kidney repair using MSC will allow the consortium to translate this knowledge into a clinical product. The consortium has delivered a technology for large scale closed system expansion of MSC based cellular products which will be translated into a clinical protocol for which an IMPD was drafted for the production of an MSC based cellular therapy for the treatment of patients with renal disease. Successful implementation of the therapy might have potential impact not only in patient care but also from a health economic and scientific viewpoint. It will impact both industry and SME development in the regenerative medicine field. An ever increasing number of individuals rely on dialysis for which the direct costs are around 50.000 euro per year. While transplantation is a cost-effective alternative to dialysis, the increasing gap between patients on the waiting list and the shortage in organs available for transplantation will lead to an increase in patients requiring dialysis over the next ten years. This constitutes a huge economic burden to society, with limited therapeutic strategies at the moment. Reducing the requirement for dialysis by even small percentages using MSC, thus would already lead to a major cost reduction in health care expenditure as shown in the cost utility analysis performed in the STELLAR project. With respect to impact on patient wellbeing, clinical application of MSC as a therapy, that can reverse chronic kidney disease is likely to have major impact on wellbeing, quality of life and exercise tolerance of patients with the prospective for societal and labour participation.STELLAR assembled high level academic institutions in Europe and Australia combining scientific expertise and knowledge on fundamental and translational research on kidney disease and MSC therapy. From a scientific point of view the collaborative efforts of the consortium had an impact on the basic understanding of the development of kidney fibrosis, and provide new inside on how to supplement renal repair. The consortium will suggest a biomarker signature to monitor the effects of cellular renal regeneration therapy. Using a high-throughput biomaterials platform the consortium searched for cues provided by kPSC to give them their specific therapeutic potential. The possibility to turn bone marrow derived MSC into a kPSC phenotype using 3D topographies was investigated. This endeavour unfortunately turned out not to be possible. The bioreactor technology further developed in the STELLAR has the potential to be marketed by partner 7.List of Websites:website: www.stellarproject.euFacebookpage:www.facebook.com/StellarStemCellsInKidneyDiseaseContact details:Scientific coordinator:Prof. Dr. AJ RabelinkDept. Internal MedicineLeiden University Medical CenterAlbinusdreef 22333 ZA LeidenThe NetherlandsE: A.J.Rabelink@lumc.nlAdministrative coordinator:Dr. B WielesDept. Immunohematology and BloodtransfusionLeiden University Medical CenterAlbinusdreef 22333 ZA LeidenThe NetherlandsP: +31715263413E: b.wieles@lumc.nl
