1. DFG-Special research area SFB Transregio 37: Micro and Nanosystems in medicine – Reconstruction of biological functions

Hannover, Aachen, Rostock
Term: 1.7.2007–30.06.2012

Scientists of the RTC are participating in three subprojects.
For more information about the overall project see: www.sfb-transregio-37.de

Subproject A4: Laser -induced Forward Transfer of Bioactive Materials

Principal Investigators:
Prof. Dr. rer. nat. Boris Chichkov, Hannover
Prof. Dr. med. G. Steinhoff, Rostock
Prof. Dr. med. Peter M. Vogt, Hannover
Associate (Rostock): Ralf Gäbel

Project goal: The Tissue Engineering of replacement tissue and organs is a central field of regenerative medicine. The optimization of cell colonization methods with stem cells is of particular interest here.
Through the modification of so-called laser-induced forward transfer (LIFT), a technology known from the industrial production of electronics, biologically active agents of many kinds as well as vital cells can be selected with the greatest purity and highest precision. Also, they can be three-dimensionally arranged in a recipient matrix.
The technology allows for the definition of tissue-specific structures and consequently for the manufacturing of highly complex tissue constructs. This technology will be applied to the generation of artificial skin replacements as well as to the cell colonization of cardiac patches for the regeneration of myocardial infracted tissue.

Subproject B2: Mikro and Nanofibre Structures

Principal Investigator:
Prof. Dr. rer. nat. Doris Klee, Aachen,
PD Dr. Nan Ma, Rostock
Associate (Rostock): PD Dr. Nan Ma

Project goal: Current developments in reconstructive medicine set high quality standards for modern scaffolds for tissue engineering which top the passive biocompatibility by far. Further prerequisites for the reconstruction of functional tissue are - beside the biodegradability - a specific surface functionality and a three-dimensional structure which is as similar as possible to the tissue to be replaced and enables vascularisation.

Subproject B5: Magnetic Polymer Nanoparticles

Principal Investigator: Prof. Dr. med. G. Steinhoff
Associates: Dr. Wenzhong Li, Evgenya Delyagina, Weiwei Wang, Yue Zhang

Project goal: The aim of the project is the development of

(i) an effective gene and drug delivery system based on magnetic nanoparticles (designated as “magnetic vectors”) and
(ii) a custom-made magnetic microsystem with a pole-row for the targeted modification of stem cells with a magnetic vector.

The magnetic vector consists of a cationic polymer which is connected to the magnetic nanoparticles. Via external magnetic fields the cationic polymer can be directly guided into individual cells. The magnetic microsystem is based on a custom-made micropole array which is implemented in a micro flow chamber in order to generate dynamic magnetic fields which guide the magnetic vectors directly into the cells.

2. Joint research project “Added Value in Regenerative Medicine – a Pioneering Approach to facilitate Translation”, Start-up of a reference- and translation center for cardiac stem cell therapy

Principal Investigator: Prof. Dr. med. G. Steinhoff
Project duration: 01.09.2008–31.12.2012

This joint research project is the basis for the foundation of the RTC. In cooperation with several partners the continued development of cardiac stem cell therapy which was initiated in Rostock will be fostered so that it can everywhere be applied as a standardised therapy of high quality to patients showing appropriate indications. This requires proof of efficacy and safety of the therapy in accordance with all relevant regulations and guidelines. This is currently being done in the frame of the clinical study PERFECT and the following research projects.

Research accompanying the clinical study PERFECT

In detail, the following topics are currently considered:
The German Heart Institute Berlin (Prof. Christoph Stamm, Andreas Bader) focuses on the characterisation of protective effects of CD133+ on ischemic/hypoxic cardiomyocytes. The co-culture experiments which have initially been performed with cord blood derived-MSC, are currently beeing transfered to human CD133. Furthermore, a method for protein-chemical characterisation (Western Blot) of post-hypoxic cardiomyocytes is being etablished.

The Hannover Medical School (PD Dr. Ingo Kutschka, Dr. Andreas Martens) studies the injection efficiency and biodistribution of stem cells after intramyocardiac injection to aim for an improvement via carrier-substances (fibrinogen). The experiments have initially been performed with iPS cell lines, and are currently being extended to human CD133+ cells.

Miltenyi-Biotec GmbH (Dr. Andreas Bosio, Dr. Ute Bissels) establishes an miRNA platform for CD133+ microarray- and realtime-data analysis. This miRNA platform is to enable the comparison of miRNA -profiles of different donors as reference with those of patient-samples from the PERFECT study. Furthermore a transgene mouse model is to be established.

Research program CD133+

The efficiency and the safety of cardiac stem cell therapy is studied using the example of CD133+ stem cells. Project targets include the improvement of the isolation of stem cell subpopulations from bone marrow. Furthermore, the role of miRNA in the stem cell differentiation as well as the effect of different genes/proteins on the recruitment of stem cells to heart will be analysed in vitro and in vivo. New methods for analysing the stem cell function need to be established for it. Three research groups of the RTC are concerned with these topics: the basic research group (genomic/proteomic),
the accompanying research group (tumor safety/animal models), and the junior research group (stem cell differentiation).

Subprojects of the basic research group (genomic/proteomic)

G1: Comparative analysies of secretomes of CD133+/CD271 stem cells derived from different human bone marrow-donations
Subproject leader: Dr. Cornelia Lux
Associate: Peter Mark
Project goal: Secreted proteins of CD133+ and CD271+ positive cells will be compared. The aim of the project is to improve the characterisation of the respective cells, and to clarify the role of cell-specific proteins for cardiac regeneration.

G2: Functional analysis of selected candidate genes or proteins in in-vitro systems
Subproject leader: PD Dr. Nan Ma
Asscociate: Dr. Dario Furlani, Dr. Lailiang Ou, Erik Pittermann, Yue Zhang
Project goal: Analysis of selected functional proteins, which impact / improve stem cell homing to heart.

G3: Evaluation of the biological significance and molecular effects of selected candidate genes and proteins in NOD-SCID animal models
Subproject leader: Dr. Jun Li
Asscociate: Marion Ludwig
Project goal: Charaterisation of CD117+AT2R+ cells — isolated from infarcted mouse hearts — and their transdifferentiation to heart cells. It is to be shown for the first time that heart cells can be generated via merging of stem cells. With this, the myocardiac regeneration could be accelerated by cell implantation.

G4: Development of a new homologous study model of bone marrow-derived CD133+ stem cells in a mouse model
Subproject leader: Peter Mark
Associate: Anita Tölk
Project goal: Murine ckit+cell-subpopulations are to be studied as an equivalent to human CD133+ cells. These murine cells will be isolated and characterised to etablish a homologous mouse model.

Subprojects of the accompanying research group (tumor safety/animal models)

B1: Comparative analysis of migration capacity of CD133+/CD34+ cells in vitro and in vivo: Setup of new examination methodologies of stem cell function
Subproject leader: Dr. Cornelia Lux
Assistant: Dr. Peter Donndorf, Michael Laupheimer, Dritan Husseini , Peter Mark
Project goal: Establishment of models to study the SDF-1-dependent migration of CD133+cells in vitro (Boyden chamber) and in vivo (Cremaster muscle model).

B2: Confirmation of tumor safety and analysis of tissue toxicity in mouse models; etablishment of investigational systems and standard parameters
Subproject leader: PD Dr. Nan Ma
Subproject goal: Identification of the tumor potential of CD133+cells for the demonstration of safe application to humans.

Subprojects of the junior research group (stem cell differentiation)

N1: Development and/or optimisation of miRNA-stem cell-microarray-technology
Subproject leader: Peter Mark
Project goal: First description of the complete miRNA expression profiles of CD133+ cells after pre-treatment or in comparison to other stem cells.

N2: Identification of microRNA-development biomarkers and expression profiles in stem cells
Subproject leader: Peter Mark
Associate: Dr. Cornelia Lux, Antje Müller
Project goal: Study of specifically regulated CD133+ cell-miRNA regarding its effect on differentiation and angiogenesis respectively. The aim is to amplify the properties of CD133+ cells.

N3: Therapeutical and /or diagnostical value of microRNA in cardiovascular diseases
Subproject leader: Dr. Wenzhong Li
Associate: Yue Zhang, Weiwei Wang
Project goal: The therapeutical effect of different CD 133+cell-miRNAs on the infarcted heart will be systematically investigated. For this, cells will be transfected with miRNA and analysed in animal model.

3. Joint research project “Development and quality management of new stem cell therapies using hematopoietic (CD133+) and mesenchymal (CD271+) stem cells to improve the regeneration efficiency of the heart”

Project duration: 01.01.2011–31.12.2013
Principal Investigator: Prof. Dr. med. G. Steinhoff
Associate: Dr. Cornelia Lux, Ralf Gäbel, Marion Ludwig, Anita Tölk

The goal of the joint research project is to develop the first generation of stem cell therapies of the diseased heart under strict scientific criteria, to gain knowlegde about the manner of effect, and to make required preparations for the market authorisation. This means that the therapy with the already applied stem cell products (CD133+-medicinal products) needs to be established as “golden standard” by application to other heart diseases and by an accompanying research program.To prepare additional therapeutical approaches, much promising stem cell subpopulations such as mesenchymal stem cells should be investigated with regarding to their potential for cardiac therapies. Furthermore, the capacity of its stem cell-effect in heart should be further improved by genetic engineering.
In addition, basic studies on regeneration mechanism should be performed and knowlegde about the impact of an application of stem cells on regeneration of the myocardium should be gained.