Cell-based regenerative medicine

Autologous Heart Tissue for Myocardial Repair

Satellite Cell Network (SatNet) - Fate Determination and Maintenance of Muscle Stem Cells

Mesenchymal stem cell mediated preconditioning of human islets

iPS and adult bone marrow cells for cardiac repair

Regenerative potential of mesenchymal stem cells (MSC)

START-MSC2; Standardization for Regenerative Therapy – Mesenchymal Stem Cells

Pluripotent Cells for Heart Therapies

Cord Blood-HEmatopoietic stem cells: Reliable Methods for ex-vivo ExpanSion

1. Objectives of funding

A lot of progress has been made in revealing the molecular mechanisms of the development of organs and tissues. Furthermore a population of hardly differentiated cells possessing a high proliferating capacity has been isolated and cultured. These cells are able to develop progenitors of a wide range of specialized cells and can build up different tissues. Naturally these precursor or stem cells are responsible for regeneration and repair processes following injuries and diseases and meanwhile have been identified in almost every tissue investigated.
On the basis of such knowledge systematic use of the body’s own regeneration potential in therapeutic approaches is becoming a distinct possibility. These new treatment strategies, covered by the term “regenerative medicine” may be applicable for any type of disease in which functions of single cell types, tissues or organs are affected.
Basic Research in this area has already been supported within the framework of the funding priority "Biological replacement of organ functions" of the Federal Government's Health Research Programme. However, considerable efforts are still needed for developing efficient methods and exploiting promising experimental approaches to clinical application.
The aim of the new measure is therefore to continue strengthening application-oriented research in the field of cell-based regenerative medicine as a follow-up to previous funding. Relevant research potential as well as the available know-how and resources are to be pooled by bringing together the best and most competent partners in interdisciplinary collaborations for the development of regenerative treatment concepts for defined clinical pictures.

2. State of the funding measure

At the beginning of 2008, the scientific community was invited to submit project applications by a public call. Until the deadline of application at 14 April 2008, 58 network proposals, comprising 232 subprojects, were submitted. In course of the project evaluation by an international review board, 15 networks comprising 57 subprojects, were selected for funding. The selected projects have started their work between March 2009 and March 2010.

3. Funded projects

a) Short description of current projects

(Sort according to project number)

Cell Therapeutic Approaches in Models of Biliary Fibrosis

Liver damage and chronic liver diseases represent a significant and severe health care problem with high medical needs since there are no efficient therapies so far. Currently, the only treatment option for the end-stage disease is liver transplantation, which is associated with severe medical complications and hampered by the scarcity of organ transplants. Preventing the progression of fibrogenesis and the revival of endogenous repair mechanisms would be beneficial for patients and reduce long-term treatment costs. Therefore, the aim of the consortium is to establish the foundations for alternative cell-based therapy approaches to human liver disease. Within the consortium, different cell sources - small hepatocytes, programmable cells of monocytic origin (PCMOs), embryonic stem cells and hepatocyte-like cells (NeoHeps) - are profoundly characterized, scaled up to an amount sufficient for transplantation and tested for their suitability to treat biliary fibrosis in animal models of liver disease.

Identification and characterization of transplanted cells in liver tissue (Subproject 2)

Evaluation of liver fibrosis scores in mice after cell transplantation based therapy (Subproject 5)

Hepatic repair from fibrosis by cell-based therapy and local EPO preconditioning (Subproject 4)

Improvement of PCMO expansion in vitro by blocking TGF-beta/activin signalling (Subproject 3)

Small hepatocytes in liver regeneration (Subproject 1)

Autologous Heart Tissue for Myocardial Repair

Cell-based therapies of myocardial diseases are emerging. By providing a new technology to construct force-generating human engineered heart tissue (hEHT), the consortium has contributed to the recent progress in the field. The translation of any cell-based cardiac repair concept is, however, hampered by restricted cardiac differentiation in embryonic stem cells (ESCs) and the paucity of autologous myocardium “repairing” cells. The consortium wants to capitalize on the cardiac-lineage inducing transcription factor MesP1 and of novel non-embryonic, potentially patient-specific pluripotent stem cells; namely spermatogonial stem cells (SSCs), induced pluripotent stem cells (iPSs), and parthenogenetic stem cells (PSCs). Forced-expression of MesP1 is used to induce cardiac lineage determination. Multipotent cardiovascular progenitors as well as cardiomyocytes from genetically naïve and “MesP1-enhanced” SSCs, iPSs, and PSCs are isolated by magnetic cell sorting using antibodies directed against VEGFR2 or a lineage restricted CD4-epitope. Similar experiments are performed with ESCs to enable direct comparisons of embryonic and adult tissue-derived pluripotent stem cells. Finally, the propensity of the different stem cell-derivatives to generate EHT in vitro and to engraft, mature and functionally integrate into native myocardium in vivo either after direct intramyocardial injection or implantation as EHT are assessed. The aim of the consortium is to advance the recently developed cardiac repair concept towards a clinical application.

Cardiac lineage determination and selection (Subproject 4)

Induced pluripotent stem cells for cardiac repair (Subproject 2)

Parthenogenetic and spermatogonial stem cells for cardiac repair (Subproject 1 and 3)

Subproject 1
Unfertilized oocytes can be chemically activated to form parthenogenetic blastocysts with an inner cell mass containing parthenogenetic stem cells (PSCs). The group could recently demonstrate that murine PSCs can give rise to bone fide cardiomyocytes in vitro and in vivo. The project therefore wants to utilize PSCs in cell-based cardiac repair. Murine and human PSCs are differentiated in embryoid bodies and subjected to magnetic activated cell sorting for VEGFR2+ derivatives. Growth and differentiation potential of the latter are analyzed to identify cardiovascular progenitors. Implantation studies in SCID-mice and in immune-suppressed pigs are performed to assess survival, maturation, and integration of murine and human PSCs as well as derivatives in vivo. Subsequently, the project wants to generate engineered heart tissue (EHT) either from multipotent PSC-derivatives or PSC-derived cardiomyocytes. PSC-EHT morphology and function are compared to genetically naïve and “MesP1-enhanced” SSC-, iPS-, and ESC-EHT. Ultimately, the project establishes an autologous mouse model of PSC-EHT-based cardiac repair and aims at providing large human PSC-EHTs as a “next step” towards a potential therapeutic application.

Subproject 3
Cell-based myocardial repair would benefit from the availability of a scalable and autologous surrogate cell source. The group recently identified multipotent adult germline stem cells (maGSCs) from mouse testis. These cells are able to differentiate into functional cardiomyocytes and vascular endothelial and smooth muscle cells in vitro. However, it is unclear, which cardiovascular cell population is transplantable and suitable for repair of the damaged heart without tumorigenicity. Therefore, protocols for differentiation of cells suitable for organ regeneration must be developed before the cells are used to define the proper therapeutic strategy in an animal model. Accordingly, the goals of the present subproject are: 1) the establishment of protocols to generate proliferating cardiovascular progenitor cells (Flk1+ in mouse and KDR+ in human) from mouse maGSCs and from human ESCs and maGSCs; 2) isolation and cultivation of proliferating cardiovascular precursors (Flk1+ in mouse and KDR+ in human); 3) differentiation of the generated Flk1/KDR+ cells into cardiomyocytes and vascular endothelial and smooth muscle cells in vitro; 4) transplantation of mouse Flk1+ cells in mouse hearts with myocardial infarction; 5) histological and functional analyses of transplanted hearts; 6) injection of mouse Flk1+ and human KDR+ cells into SCID-beige mice to study their tumorigenicity. The study intends to help to develop novel treatment strategies for heart failure.

Satellite Cell Network (SatNet) - Fate Determination and Maintenance of Muscle Stem Cells

Healthy adult skeletal muscle has an impressive ability to regenerate, which it does on a daily basis to maintain muscle mass, or in response to injury in order to replace damaged muscle fibres. In congenital and age-related muscle disease, the regenerative potential of muscle stem cells does not suffice to repair or maintain skeletal muscle, due to impaired differentiation, senescence and/or depletion of the satellite cell pool. The consortium investigates the molecular mechanisms and networks that determine satellite cell fate and allow stem cell maintenance and self-renewal. The respective knowledge is a precondition for stimulation of the endogenous satellite cell-mediated repair or for the expansion of cultured satellite cells for a cell-based therapy of muscle disease.

Regenerative potential of muscle stem cells (Subproject 4)

Muscle stem cell determination/maintenance (Subproject 2)

RBP-J/Notch and satellite cell fate (Subproject 1) and A transcriptional network defining stemness (Subproject 3)

Mesenchymal stem cell mediated preconditioning of human islets

MSZ zur Induktion der Inselzelltoleranz im humanisierten Diabetes Modell

MSC-Migration als ein kritischer Schritt in der Toleranz von Allotransplantaten

Rolle der Indoleamin-2.3-dioxygenase (IDO) bei der Interaktion von MSC mit Pathogenen und mit T-Zellen

MSZ-vermittelte Konditionierung humaner Inseln

Verbesserung von Inselzellüberleben und Inselzellregeneration durch MSC - Therapie

iPS and adult bone marrow cells for cardiac repair

Cardiac cell therapy is a promising approach to treat heart disease. Autologous bone marrow cells have some beneficial effects on cardiac function after myocardial infarct, but their cardiac persistence and ability to differentiate into cardiomyocytes (CM) are limited. In contrast, neonatal or fetal CM and CM derived from embryonic stem cells (ES-CM) survive and remain in loco, electrically couple to host CM and support contractility after intramyocardial injection.
The aim of our consortium is to drive cardiac cell therapy one step closer to the edge of clinical application. The following hypotheses will be tested:
1) CM can be obtained from the very promising source of induced pluripotent stem (iPS) cells in sufficient purity to allow cardiac regeneration without tumor formation.
2) iPS cell derived CM (iPS-CM) couple structurally and electrically to host CM in vitro (subproject II) and in vivo after myocardial infarction.
3) Engraftment of iPS-CM is enhanced by optimizing cell application modalities and pre-treatment of cells.
4) iPS-CM improve cardiac function in different states of disease and after different modes of cell application.
5) iPS-CM for cardiac repair are safe and do not lead to tumor formation even when exposed to physical stress.
In addition, we will evaluate the hypothesis that the combination of adult mesenchymal bone marrow cells (MSC) with iPS-CM is an advantageous approach to improve cell deposit (MSC for better adherence), cell survival (MSC for reduction of cell death), electrical and functional integration (MSC for cell coupling), and overall or regional cardiac function without inducing tumor formation. The results of this project will provide important data on the therapeutic potential of IPS-CM and their optimal use. Fetal CM and ES-CM are studied for comparison.

Effect of MSC on tumorigenic potential of iPS-CM

iPS cell-derived cardiomyocytes, 2) in vitro analysis of integration, 3) enhancing iPS-CM engraftment, 4) electrical integration of iPS-CM in vivo and 5) iPS-CM and MSC: in vivo evaluation

Regenerative potential of mesenchymal stem cells (MSC)

We would like to use the increasing knowledge of the molecular regulation of MSC proliferation and differentiation to specifically explore the potential of human MSC. We will focus on the immunogenicity and the immunomodulatory effects of autologous vs. allogeneic MSCs and their modulation by clinically relevant immunomodulatory agents and extracellular matrices to define the most promising tools for regenerative therapy. This aspect will be of relevance for testing the possibility to improve the engraftment and survival of pancreatic islet grafts. The role of MSC within various stem cell niches will be studied by a molecular analytical project in which signalling pathways activated in or by MSC (e.g. BMP, Hedgehog, Wnts) can be detected by a fluorescence based reporter assay. Since the proliferation and biological function of MSC can be modified by the way of isolation and the culture carrier used for expansion, experiments will focus on the regenerative and trophic effects of purified MSC subsets cultured/expanded on bioactive polymer scaffolds. We like to address whether the osteogenic potential of MSC can be enhanced by the use of bioactive substrates. Beside this we investigate the in-vivo regeneration of bone mediated by mesenchymal stem cells in a zebra-fish model.

Materials to control MSC in culture

1) Immunosuppressive capacity and immunogenicity of MSC, 2) MSC in islet transplantation, 3) MSC in zebrafish fin regeneration, 4) imaging Mesenchymal Stem Cell Signalling and 5) materials to control MSC in culture

START-MSC2; Standardization for Regenerative Therapy – Mesenchymal Stem Cells

The lack of common standards for preparations of stem cells and of differentiated cells derived thereof has remained a major obstacle for progress in cell-based therapy. For mesenchymal stem cells (MSC), the quality of preparations from different laboratories varies tremendously. Within the past 3 years this consortium has established optimized protocols for preparation of MSC, developed tools and a catalogue of markers for precise characterization, defined the impact of cellular senescence on their potential, and studied their differentiation potentials in vitro, in a blastocyst injection and in animal models. We have demonstrated that albeit optimized MSC preparations were able to differentiate into bone, cartilage and adipose tissues, there is no evidence that they can differentiate into liver or cardiac cells. However, the induction of pluripotency by introducing defined genetic factors into somatic cells has opened new horizons. Considering these developments, we propose in Start-MSC2 to verify and establish guidelines for quality control of optimized MSC preparations and compare them with genetically manipulated counterparts, such as iP-MSC. We will pursue the following goals: 1. Development of standard procedures and guidelines for the safe and efficient production of human MSC under GMP-conditions. 2. Application of optimized and standardized MSC-preparations as a surrogate niche-model for hematopoietic stem cells. 3. Impact of replicative senescence upon gene expression, junctional complexes and functional properties of MSC.  4. Identification of differentiation mechanisms and development of reprogrammed MSC with induced pluripotency.

In vitro and in vivo characterization of hepatic progenitor cells derived from standardized pluripotent/multipotent human MSC populations

MSC developmental potential

Molecular Differentiation Markers for Cardiomyogenesis, Hepatocytogenesis, Adipogenesis and Interstitial Cells

Differentiation Mechanisms & Reprogrammed MSC

Gene and protein profiles of human MSC subsets

Towards GMP-Processing: Banking, Characterization and GMP-Manufacturing of MSC

Pluripotent Cells for Heart Therapies

The socioeconomic burden of cardiovascular diseases and the principal feasibility of therapeutic cell applications in the treatment of acute heart infarctions give important reasons for studying patient-derived stem cells as possible sources for transplantation approaches. During the current first funding phase we aimed at using fusion hybrid cells to obtain patient-derived pluripotent stem cells that can be used in regenerative cardiac medicine. However, we did not succeed in generating true diploid patient-derived cells from the tetraploid fusion hybrids. Nevertheless, we provided important new insides in the molecular mechanisms of somatic cell reprogramming and in the characteristics of fusion hybrids during mesodermal / cardiac cell differentiation. Moreover, we established an active, interacting consortium of renowned research groups with comprehensive expertise in the field of stem cell-based therapeutic applications for cardiac diseases. Amongst the recent achievement of the Max Planck Institute for Molecular Biomedicine in Muenster (MPI-MBM) the generation of truly pluripotent stem cells, derived from murine testis biopsies, offers a promising new cell resource for patient-specific therapeutic approaches. Therefore, we propose to analyze the potential of these pluripotent germline stem cells (pGSC) with respect to their cardiac differentiation capacity, their immunological characteristics, and their significance as source for cardiac cell transplants. Furthermore, we aim to generate porcine and human pGSC for the evaluation in pre-clinical large animal models of the pig to finally provide a cell therapeutic strategy for future phase-I clinical trials.

Transplantation of pGSC-derived cardiac cells

pGSC-CM: Immunogenetic and functional properties

Cardiac in vitro differentiation of pGSC

Evaluation of porcine pGSCs

Pluripotent germline stem cells

Cord Blood-HEmatopoietic stem cells: Reliable Methods for ex-vivo ExpanSion

Lifelong blood production depends on haematopoietic stem cells (HSCs) and their ability to self-renew and to differentiate. Cord blood (CB) banking is continually increasing due to the superior properties of CB compared to adult HSC. However our inability to expand HSCs renders insufficient stem cell numbers, a major constraint in many settings of CB-HSC transplantation. Despite optimization of isolation and processing techniques this restricts CB-HSC transplantation mainly to paediatric patients. New methods that generate sufficient numbers of HSCs from limited input cells are needed to make CB-HSCs available to adult patients and amenable to advanced cell and gene therapy approaches in regenerative medicine. Therefore, the aim of this consortium is to open CB-HSCs to new therapeutic applications by developing controlled strategies for expansion and transplantation. Specifically we plan to apply novel growth factor cocktails, nano-structured 3D surfaces, modifications of inhibitory pathways and epigenotype and specific stroma environments in order to expand and regulate HSCs ex vivo. The first clinical application of novel strategies developed by us is in the context of allogeneic CB-HSCs transplantation for elderly patients suffering from haematopoietic disorders. Overall goal: to broaden the therapeutic application of CB-HSCs by developing robust means that allow significant HSC expansion and better engraftment. Specific aims: 1) to develop rational and robust means of ex vivo CB-HSC expansion: by novel growth factor cocktails, nano-structured 3D surfaces, modification of inhibitory pathways, induced epigenetic modifications and by specific stroma environments; 2) development of clinically applicable standard operating procedures for CB-HSC expansion using CD34+ cells isolated from umbilical cord blood; 3) eludicate molecular pathways and intercellular networks operating in HSC ex vivo expansion cultures; 4) exploring genetic, epigenetic and functional integrity of expanded cells in vivo.

Expansion of CB-HSCs with human MSCs

Epigenetic characterization of CB-HSCs and SP6: Coordination and Administration

Biomaterial scaffolds for CB-HSC expansion

Pathway discovery and protocol development and SP5: Clinical application of CB-HSCs

b) Expired Projects