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What are stem cells?
Stem Cell Therapies- State of the art and future perspectives
What is cardiac stem cell therapy?
How do we generate stem cell for the therapy? 

 
 

What are stem cells?

“Stem cells” are defined as such cells which are able to continually renew themselves, thus to produce daughter cells and, additionally, to differentiate into different cell types.
Depending on the particular class of stem cells they have the potential to reproduce all cells of the body or only defined cell types. The former ability particularly applies to embryonic stem cells and the latter ability applies to adult stem cells. The ontogenetic age of stem cells hence comes along with their differentiation potential: the ontogenetically youngest stem cells are embryonic stem cells from which later the somatic stem and progenitor cells emerge.

Embryonic stem cells (ES cells) have the ability to differentiate into cells of all three germ layers (endoderm, ectoderm, mesoderm) and into germline cells both in vivo and in vitro. Accordingly, they are able to differentiate into any type of cells of the body, and therefore are termed pluripotent.  ES cells are derived from the inner cell mass (ICM, also called  embryoblast) of the blastocyst after an in vitro fertilization during the embryonic phase. By this means, they can be established as cell lines  in vitro.

Since the preparation of ES cells comes along with the damage of embryos research on human embryonic stem cells (mostly abbreviated as hES cells) caused an intense ethical debate within the society which is even continuing today. Therefore, the legislative body strictly regulates corresponding research in the German Stem Cell Act (Deutsches Stammzellgesetz, StZG) and allows research only under strict conditions (Key date regulation). In Germany the preparation of hES cells is illegal according the Embryo Protection Act (Embryonenschutzgesetz (EschG)).

Adult stem cells
In contrast to the embryonic stem cells deriving from an early stage embryo there are still stem cells in large number of tissues in adult bodies which have, however, very  limited differentiation potential (“multipotent”).  These cells are able to give rise to specialized cells over the entire life time of the organism.

These adult, multipotent stem cells are particularly found in tissues such as bone marrow, skin, adipose tissue as well as in the brain, liver, salivary gland, root of a hair and elsewhere.  In comparison to embryonic stem cells they typically have a  reduced self-renewal capacity and clearly limited differentiation potential. The differentiation of certain adult stem cell types into lineages of all three germ layers (“transdifferentiation”) has been described in various studies but remains controversial.
Adult stem cells are found in each individual such they are applied as autologous cells for the development of therapeutic approaches. As autologous cells their rejection by the immune system is much more unlikely as compared to ES cells. In addition, it is being assumed that tumour genetic alteration of adult stem cells is rather improbable. Until now, tumor genetic alterations as a consequence of the clinical application of adult stem cells has not been observed.

Induced pluripotent stem cells (iPS)
In 2006, Kazutoshi Takahashi und Shinya Yamanaka were the first to succeed in the programming of mouse adult differentiated cells into so called "induced pluripotent stem cells" (iPS). In 2007 they managed to do the same with corresponding human cells. For this purpose, the four transcription factor genes Oct4, Sox2, c-Myc and Klf4 were inserted into the cells by means of viruses. This reprogrammed the somatic cells to a pluripotent state. The  researchers were able to generate differentiated cells such as myocardiocytes and neurocytes in the petri dish from the artificially reprogramed stem cells – analogously to the embryonic stem cells.  For this pioneering discovery, Yamanaka was awarded the Noble Prize in Medicine in 2012.
Like the adult stem cells iPS cells can also be generated autologously and can therefore also be specifically produced for a particular patient.  Recently, the first clinical trial with patients treated with iPS cells has been started. The aim of this trial is the treatment of macular degeneration. It needs to be clarified  whether the iPS cells are able to effectively meet the requirements of safety for the therapeutic application in the future since at the moment a tumourigenetic alteration can not be excluded in all pluripotent cell types.
The latter is expected for the direct programming of differentiated cells -such as fibroblasts- into myocardiocytes. A number of fundamental publications have evolved from the primary work of the research group of D. Srivastava (2010) in this highly topical research field. The principle of the method is based on the programming of the cells into a new cell type by avoiding the pluripotent status of an iPS cell. Currently, our group is working on the development of novel  programming strategies which avoid viral vectors which have been used to date.

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Stem Cell Therapies- State of the art and future perspectives

Self-renewal – also called regeneration – is a process which the body is able to maintain up to a determined level. According to the latest research results,  organs such as bone marrow, liver, and intestine have a high regeneration potential by a continuing cell renewal. This process is based on the resident stem cells of these organs.  Other organs such as the heart and the brain are less regeneration-capable.
By means of stem cell therapies damaged cells are to be replaced by applied stem cells. This means, that stem cells differentiate in vivo to target cells or they stimulate the neighbouring tissue  to generate functioning cells (paracrirne effects). Because of the predominant legal situation in terms of the  application of  embryonic stem cells in Germany, so far, the research focuses on the adult stem cell therapy in the clinical field. The breakthrough for the  first stem cell therapy was already in 1969 -  i.e., the bone marrow transplantation for the treatment of leukemia.

To close chronic wounds, for example, outer root sheath stem cells have been used to generate skin cells. The resulting skin flaps are then put on the wound of the patient for engraftment. In the field of cardiovascular diseases where mostly autologous bone marrow-derived stem cells  are applied notable promising results have been gained. In this area the approaches are under way to standardized therapeutic application.
A general reimbursement for these therapies by the compulsory healthy insurance does not exist yet but reimbursement in individual cases is possible. To date, only few products received the approval as an “Advanced Therapy Medicinal Product” in Europe and in Germany, respectively. Among them are some products for the treatment of damaged articular cartilage and a method for urethral reconstruction with oral mucosa cells.
 

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What is cardiac stem cell therapy?

In Germany, every year about 280.000 people suffer from heart infarction caused by blockage of one or several coronary blood vessels. The neighboring myocardial tissue then can not sufficiently  be supplied with oxygen and nutrients and may finally die. The heart is hardly able to regenerate or to repair the damaged tissue by itself.
The currently applied common treatment of myocardial infarction by by-pass surgery  or stent implantation improves the quality of life and the life time of the patients. However, the original effectiveness of the heart can not be restored. Therefore most of the patients suffer from chronic sequelae. In case of a severely damaged myocardium an organ transplantation can not be avoid. Researchers have recently succeeded to discover cardiac stem cells within the myocardium, however their cell number is very low such that they are not able to regenerate the damaged myocardium after severe diseases such as after a myocardial infarction. The  “regrowing heart” is currently still a vision. Various research approaches are being pursued to reach this goal. For instance, it has been shown that mouse and human embryonic stem cells were able to differentiate to myocardiocytes after treatment with growth factors. Hence, this implies that one day these cells could potentially replace the dead myocardiocytes.
One innovative treatment approach which has successfully passed a clinical study and is  already being used for therapeutic application is the stem cell therapy with autologous bone marrow-derived stem cells. These cells have intramyocardially been applied to the damaged area of the heart to replace and regenerate the damaged target tissue.
At the Clinic and Policlinic for Cardiac Surgery of Rostock a therapy has been developed to treat patients with autologous stem cells additionally to a by-pass surgery after a myocardial infarction. For this purpose, defined stem cells are isolated from the bone marrow of the patient and are intramyocardially injected into the damaged area of the myocardium during the by-pass surgery. The stem cell population used for the “Rostock Therapy” is identified by the marker CD133 on the surface of the cells.

 

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How do we generate stem cells for the therapy?

At the RTC  all applied stem cell preparations have been manufactured according to the actual pharmaceutical law. This makes sure that the requirements of the “Pharmaceuticals and Active Agent Manufacturing Ordinance” (German: “Arzneimittel- und Wirkstoffherstellungsverordnung” (AMWHV)) for the application of “Good Manufacturing Practice” (GMP) and for the manufacture of products of human origin according to the “Codes of Good Practice” (Gute Fachliche Praxis, GFP) are met. Currently, all of our stem cell products are being produced by a manufacturer with the corresponding authorization. 

Reduced production times by widely automated processes stimulated us to focus on „Point of Care“(PoC)-manufacturing (on-site patient care) of stem cell products. By means of such GMP-compatible procedures we will manufacture such medicinal products on our own by “Point of Care” practice in those cases which are advantageous. For this purpose we have installed production sites close to the operation halls in agreement with GMP requirements and we have established appropriate measures for quality assurance. In the near future we will apply to receive authorization for manufacture from the local authorities.

 

CD133+ stem cell isolation by CliniMACS of Miltenyi Biotec as an example for the principle of stem cell isolation

For the CD133+ cell isolation autologous bone marrow from the iliac crest of the patient serves as the primary material. The bone marrow is being removed and collected during the surgery procedure.  After the centrifugation of plasma and erythrocytes the cell separation of the selected stem cell population is performed by means of specific antibody-coated magnetic particles. These CD133 antibodies only bind to such stem cells which express the marker CD133. During the passage through a magnetic column only such magnetic particles which are bound to the antibody-CD133+stem cell complex adhere to the magnetic column. In the next step the cells can be isolated after separation from the residual cell suspension. The isolated CD133+ stem cell preparation is collected in plasma or serum, and committed to the physician for the therapy after successful evaluation and approval.

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How is the status of clinical development at the RTC?

PERFECT – first Phase III stem cell trail for heart regeneration

Autologous stem cell products for the cardiac application have been classified as “Advanced Therapy Medicinal Products” and are underlying the strict regulations of the  Medicines Act. For this reason, quality, efficacy and safety for the application in human beings have to be proven before putting such a product on the market. During the period between 2001 –2005 clinical Phase I/II trials have already been performed in Rostock for the proof of safety and efficacy of the intramyocardial therapy with bone marrow-derived CD133 stem cells after myocardial infarction parallel to a bypass surgery.

With these trials we were able to demonstrate the safety of the therapy and a first indication of a significant improvement of the heart pump function. After that, in 2009, the first randomised double-blinded multicentric clinical Phase III trial PERFECT has been initiated in Rostock. With this  trial proof of safety and efficacy should systematically be provided. The trial in which six well-known heart centres in Germany are involved is performed according to the internationally approved requirements of “Good Clinical Practice” (GCP).

Research program of the PERFECT trial

In collaboration with the German Heart Centre Berlin, the Hannover Medical School and the company Miltenyi Biotec GmbH we conduct extensive research on the mode of action of the cardiac stem cell therapy with CD133+stem cells as part of the accompanying research program of the PERFECT trial. The aim is to identify the responsible factors for the efficacy of the cardiac stem cell therapy. The research program addresses issues such as:
 
•    Quality analysis of CD133+ stem cells
•    Analysis of surface markers of the CD133+ stem cells
•    Analysis of stem cell recruiting and functioning at different times
•    Analysis of angiogenesis in vitro and in vivo
•    Analysis of miRNA as biomarker in peripheral blood and CD133+ stem cells
•    Analysis of biodistribution

After unblinding the trial PERFECT, correlation analysis with the collected data should be conducted.

Patient care

Since 2001 almost 200 patients have been treated with stem cells at the Clinic and Policlinic for Cardiac Surgery of Rostock – an innovative therapy which is already accepted by the health insurances. By now the therapy is available for patients with the following disease symptoms:

•    after a myocardial infarction, and when a bypass surgery is needed
•    a severe reduction of pump function of the heart, proven by MRT analysis
•    heart failure as sequelae of an ischemic heart disease, and when the damaged cardiac tissue can be       clearly localised.

After the surgery the patients benefit from an extensive follow-up examination and their data will be pseudonymously recorded in a register for their entire lifetime.

Stem cell Register 

The RTC has established a register for long-term follow-up of heart patients who have been treated with stem cells. In the register the data of all patients are recorded who have been treated with stem cells in Rostock in the frame of their therapy or in the frame of clinical trials. These data are used to ensure the safety of the application of stem cell products in the cardiovascular field (therapy vigilance).

The systematic analysis of long-term data has been conducted for the establishment of a “Good Vigilance Practice” (GVP)-monitoring with which new insights in terms of long term efficacy and safety of novel therapies can be found. Based on these data we aim to formulate suggestions for standardization and quality assurance which may then be used for the formulation of guidelines for stem cell therapy of cardiovascular diseases. In the very first place, however, these analyses are of prime importance for the safety of our patients and for the optimization of our therapies.
The register is web based such that other centres may easily join it. In the frame of the PERFECT trial data from patients from all six participating centres of the „competence liaison“ will be recorded in the Register in the frame of GVP.
In the future, the RTC will more focus on the field of vigilance and health care research and try to initiate a closer communication with authorities, health insurances and other partners

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Legal The RTC is supported by the BMBF and the State Mecklenburg-Western Pomerania using EU Structural Funds.