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.


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

Principal investigator: Prof. Dr. Wolfram-Hubertus Zimmermann
Georg-August-Universität Göttingen, Universitätsmedizin Göttingen, Zentrum Pharmakologie und Toxikologie, Abt. Pharmakologie, Robert-Koch-Str. 40, 37075 Göttingen

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.


Induced pluripotent stem cells for cardiac repair (Subproject 2)

Principal investigator: Prof. Dr. Ulrich Martin
Medizinische Hochschule Hannover, Abt. HTG-Chirugie/LEBAO, Carl-Neuberg-Str. , 30625 Hannover
 
The goal of this subproject is the development of engineered heart tissue (EHT) based on murine and human iPS-derived cardiomyocytes (CM). The working plan forsees the following steps: a) Derivation of mouse and human cardiovascular progenitors (CVPCs) and cardiomyocytes (CMs) from iPS (embryoid bodies and single cell differentiation); b) characterization of iPS-derived CVPCs and CMs via qRT-PCR, immunostaining, MEA, measurement of intracellular calcium); c) lentiviral transduction to generate selectable iPS/PSC/SSC/ESC; d) lentiviral transduction to express Mesp1 in iPS/PSC/SSC/ESC; e) generation and characterization of iPS-EHT via EM, confocal LSM, force measurements, electrophysiology; e) implantation of murine and human iPS derivatives in healthy and infarcted mice and pigs (analysis of cell survival and integration via immunohistology, functional analyses including MRT and pressure-volume catheter); f) implantation of human EHT in healthy and infarcted mice (analysis as described above); g) allocation of murine and human iPS to other subprojects. After the development of the methodology, it is planned to continue with the development of a vascularised autologous contractile myocardial patch together with Corlife GbR, Hannover.


Cardiac lineage determination and selection (Subproject 4)

Principal investigator:  Prof. Dr. Wolfgang-Michael Franz
Ludwig-Maximilians-Universität München, Klinikum der Universität München, LIFE-Zentrum - Laser-Forschungslabor, Marchioninistr. 23, 81377 München
 
Stem cell based cardiac repair will require sufficient yields of cardiac cells for transplantation and tissue engineering. MesP1, the earliest known cardiovascular marker is sufficient to induce cardiogenesis in murine ES cells as recently shown by us. Therefore this subproject aims at MesP1 based “Cardiovascular Forward Programming” of various pluripotent cell types (1. iPS, 2. spermatogonial stem cells, 3. parthenogenic stem cells). In addition to directed differentiation, high-yield isolation of cardiovascular progenitors derived from the same cellular sources via magnetic cell sorting (MACS) using a MesP1-promoter driven CD4 surface marker is pursued. The purified cardiovascular progenitor cells as well as the preprogrammed cardiovascular cells are used for cell transplantation as well as tissue engineering approaches. In addition, biochemical analyses and global whole genome expression array data will supply important novel information on specific intrinsic as well as extrinsic factors, surface markers and pathways involved in cardiogenesis.


Source: http://www.gesundheitsforschung-bmbf.de/en/1195.php (14.04.2010)