Petra Wolint

Injectable living marrow stromal cell-based autologous tissue engineered heart valves: first experiences with a one-step intervention in primates

AIMS A living heart valve with regeneration capacity based on autologous cells and minimally invasive implantation technology would represent a substantial improvement upon contemporary heart valve prostheses. This study investigates the feasibility of injectable, marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) generated and implanted in a one-step intervention in non-human primates. METHODS AND RESULTS Trileaflet heart valves were fabricated from non-woven biodegradable synthetic composite scaffolds and integrated into self-expanding nitinol stents. During the same intervention autologous bone marrow-derived mononuclear cells were harvested, seeded onto the scaffold matrix, and implanted transapically as pulmonary valve replacements into non-human primates (n = 6). The transapical implantations were successful in all animals and the overall procedure time from cell harvest to TEHV implantation was 118 ± 17 min. In vivo functionality assessed by echocardiography revealed preserved valvular structures and adequate functionality up to 4 weeks post implantation. Substantial cellular remodelling and in-growth into the scaffold materials resulted in layered, endothelialized tissues as visualized by histology and immunohistochemistry. Biomechanical analysis showed non-linear stress-strain curves of the leaflets, indicating replacement of the initial biodegradable matrix by living tissue. CONCLUSION Here, we provide a novel concept demonstrating that heart valve tissue engineering based on a minimally invasive technique for both cell harvest and valve delivery as a one-step intervention is feasible in non-human primates. This innovative approach may overcome the limitations of contemporary surgical and interventional bioprosthetic heart valve prostheses.

Stem Cell–Based Transcatheter Aortic Valve Implantation : First Experiences in a Pre-Clinical Model

OBJECTIVES This study sought to investigate the combination of transcatheter aortic valve implantation and a novel concept of stem cell-based, tissue-engineered heart valves (TEHV) comprising minimally invasive techniques for both cell harvest and valve delivery. BACKGROUND TAVI represents an emerging technology for the treatment of aortic valve disease. The used bioprostheses are inherently prone to calcific degeneration and recent evidence suggests even accelerated degeneration resulting from structural damage due to the crimping procedures. An autologous, living heart valve prosthesis with regeneration and repair capacities would overcome such limitations. METHODS Within a 1-step intervention, trileaflet TEHV, generated from biodegradable synthetic scaffolds, were integrated into self-expanding nitinol stents, seeded with autologous bone marrow mononuclear cells, crimped and transapically delivered into adult sheep (n = 12). Planned follow-up was 4 h (Group A, n = 4), 48 h (Group B, n = 5) or 1 and 2 weeks (Group C, n = 3). TEHV functionality was assessed by fluoroscopy, echocardiography, and computed tomography. Post-mortem analysis was performed using histology, extracellular matrix analysis, and electron microscopy. RESULTS Transapical implantation of TEHV was successful in all animals (n = 12). Follow-up was complete in all animals of Group A, three-fifths of Group B, and two-thirds of Group C (1 week, n = 1; 2 weeks, n = 1). Fluoroscopy and echocardiography displayed TEHV functionality demonstrating adequate leaflet mobility and coaptation. TEHV showed intact leaflet structures with well-defined cusps without signs of thrombus formation or structural damage. Histology and extracellular matrix displayed a high cellularity indicative for an early cellular remodeling and in-growth after 2 weeks. CONCLUSIONS We demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic valve position within a 1-step intervention. Its long-term functionality proven, a stem cell-based TEHV approach may represent a next-generation heart valve concept.

Human stem cell-based three-dimensional microtissues for advanced cardiac cell therapies

Cardiac stem cell therapy has been proposed as a therapy option to treat the diseased myocardium. However, the low retention rate of transplanted single-cell suspensions remains a major issue of current therapy strategies. Therefore, the concept of scaffold-free cellular self-assembly into three-dimensional microtissues (3D-MTs) prior to transplantation may be beneficial to enhance retention and survival. We compared clinically relevant, human stem cell sources for their ability to generate 3D-MTs with particular regards to formation characteristics, proliferation-activity, viability and extracellular-matrix production. Single-cell suspensions of human bone marrow- and adipose tissue-derived mesenchymal stem cells (hBMMSCs and hATMSCs), Isl1(+) cardiac progenitors derived from human embryonic stem cells (hESC-Isl1(+) cells), and undifferentiated human induced pluripotent cells (hiPSCs) were characterized before to generate 3D-MTs using a hanging-drop culture. Besides the principal feasibility of cell-specific 3D-MT formation, a detailed head-to-head comparison between cell sources was performed using histology, immunocyto- and histo-chemistry as well as flow cytometry. Round-oval shaped and uniform 3D-MTs could be successfully generated from all cell types starting with a loose formation within the first 24 h that fully stabilized after 3 days and resulting in a mean 3D-MT diameter of 194.56 ± 18.01 μm (hBMMSCs), 194.56 ± 16.30 μm (hATMSCs), 159.73 ± 19.20 μm (hESC-Isl1(+) cells) and 120.95 ± 7.97 μm (hiPSCs). While all 3D-MTs showed a homogenous cell distribution, hiPSC-derived 3D-MTs displayed a compact cell formation primarily located at the outer margin. hESC-Isl1(+) and hiPSC-derived 3D-MTs maintained their proliferation-activity which was rather limited in the MSC-based 3D-MTs. All four 3D-MT types revealed a comparable viability in excess of 70% and showed a cell-specific expression profile being comparable to their single-cell counterparts. Extracellular matrix (ECM) production during 3D-MT formation was observed for all cell-specific 3D-MTs, with hiPSC-derived 3D-MTs being the fastest one. Interestingly, ECM distribution was homogenous for hATMSC- and hiPSC-based 3D-MTs, while it appeared to be primarily concentrated within in the center of hESC-Isl1(+) and hBMMSC-based 3D-MTs. The results of this head-to-head comparative study indicated that 3D-MTs can be successfully generated from hESC-derived Isl1(+) cells, hiPSCs and MSC lines upon hanging drop culture. Cell-specific 3D-MTs displayed sufficient viability and instant ECM formation. The concept of 3D-MT in vitro generation prior to cell transplantation may represent a promising delivery format for future strategies to enhance cellular engraftment and survival.

Transcatheter aortic valve implantation using anatomically oriented, marrow stromal cell-based, stented, tissue-engineered heart valves: technical considerations and implications for translational cell-based heart valve concepts

OBJECTIVES While transcatheter aortic valve implantation (TAVI) has rapidly evolved for the treatment of aortic valve disease, the currently used bioprostheses are prone to continuous calcific degeneration. Thus, autologous, cell-based, living, tissue-engineered heart valves (TEHVs) with regeneration potential have been suggested to overcome these limitations. We investigate the technical feasibility of combining the concept of TEHV with transapical implantation technology using a state-of-the-art transcatheter delivery system facilitating the exact anatomical position in the systemic circulation. METHODS Trileaflet TEHVs fabricated from biodegradable synthetic scaffolds were sewn onto self-expanding Nitinol stents seeded with autologous marrow stromal cells, crimped and transapically delivered into the orthotopic aortic valve position of adult sheep (n = 4) using the JenaValve transapical TAVI System (JenaValve, Munich, Germany). Delivery, positioning and functionality were assessed by angiography and echocardiography before the TEHV underwent post-mortem gross examination. For three-dimensional reconstruction of the stent position of the anatomically oriented system, a computed tomography analysis was performed post-mortem. RESULTS Anatomically oriented, transapical delivery of marrow stromal cell-based TEHV into the orthotopic aortic valve position was successful in all animals (n = 4), with a duration from cell harvest to TEHV implantation of 101 ± 6 min. Fluoroscopy and echocardiography displayed sufficient positioning, thereby entirely excluding the native leaflets. There were no signs of coronary obstruction. All TEHV tolerated the loading pressure of the systemic circulation and no acute ruptures occurred. Animals displayed intact and mobile leaflets with an adequate functionality. The mean transvalvular gradient was 7.8 ± 0.9 mmHg, and the mean effective orifice area was 1.73 ± 0.02 cm(2). Paravalvular leakage was present in two animals, and central aortic regurgitation due to a single-leaflet prolapse was detected in two, which was primarily related to the leaflet design. No stent dislocation, migration or affection of the mitral valve was observed. CONCLUSIONS For the first time, we demonstrate the technical feasibility of a transapical TEHV delivery into the aortic valve position using a commercially available and clinically applied transapical implantation system that allows for exact anatomical positioning. Our data indicate that the combination of TEHV and a state-of-the-art transapical delivery system is feasible, representing an important step towards translational, transcatheter-based TEHV concepts.

Transcatheter based electromechanical mapping guided intramyocardial transplantation and in vivo tracking of human stem cell based three dimensional microtissues in the porcine heart

Stem cells have been repeatedly suggested for cardiac regeneration after myocardial infarction (MI). However, the low retention rate of single cell suspensions limits the efficacy of current therapy concepts so far. Taking advantage of three dimensional (3D) cellular self-assembly prior to transplantation may be beneficial to overcome these limitations. In this pilot study we investigate the principal feasibility of intramyocardial delivery of in-vitro generated stem cell-based 3D microtissues (3D-MTs) in a porcine model. 3D-MTs were generated from iron-oxide (MPIO) labeled human adipose-tissue derived mesenchymal stem cells (ATMSCs) using a modified hanging-drop method. Nine pigs (33 ± 2 kg) comprising seven healthy ones and two with chronic MI in the left ventricle (LV) anterior wall were included. The pigs underwent intramyocardial transplantation of 16 × 10(3) 3D-MTs (1250 cells/MT; accounting for 2 × 10(7) single ATMSCs) into the anterior wall of the healthy pigs (n = 7)/the MI border zone of the infarcted (n = 2) of the LV using a 3D NOGA electromechanical mapping guided, transcatheter based approach. Clinical follow-up (FU) was performed for up to five weeks and in-vivo cell-tracking was performed using serial magnet resonance imaging (MRI). Thereafter, the hearts were harvested and assessed by PCR and immunohistochemistry. Intramyocardial transplantation of human ATMSC based 3D-MTs was successful in eight animals (88.8%) while one pig (without MI) died during the electromechanical mapping due to sudden cardiac-arrest. During FU, no arrhythmogenic, embolic or neurological events occurred in the treated pigs. Serial MRI confirmed the intramyocardial presence of the 3D-MTs by detection of the intracellular iron-oxide MPIOs during FU. Intramyocardial retention of 3D-MTs was confirmed by PCR analysis and was further verified on histology and immunohistochemical analysis. The 3D-MTs appeared to be viable, integrated and showed an intact micro architecture. We demonstrate the principal feasibility and safety of intramyocardial transplantation of in-vitro generated stem cell-based 3D-MTs. Multimodal cell-tracking strategies comprising advanced imaging and in-vitro tools allow for in-vivo monitoring and post-mortem analysis of transplanted 3D-MTs. The concept of 3D cellular self-assembly represents a promising application format as a next generation technology for cell-based myocardial regeneration.

Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment.

More people die annually from cardiovascular diseases than from any other cause. In particular, patients who suffer from myocardial infarction may be affected by ongoing adverse remodeling processes of the heart that may ultimately lead to heart failure. The introduction of stem and progenitor cell-based applications has raised substantial hope for reversing these processes and inducing cardiac regeneration. However, current stem cell therapies using single-cell suspensions have failed to demonstrate long-lasting efficacy due to the overall low retention rate after cell delivery to the myocardium. To overcome this obstacle, the concept of 3D cell culture techniques has been proposed to enhance therapeutic efficacy and cell engraftment based on the simulation of an in vivo-like microenvironment. Of great interest is the use of so-called microtissues or spheroids, which have evolved from their traditional role as in vitro models to their novel role as therapeutic agents. This review will provide an overview of the therapeutic potential of microtissues by addressing primarily cardiovascular regeneration. It will accentuate their advantages compared to other regenerative approaches and summarize the methods for generating clinically applicable microtissues. In addition, this review will illustrate the unique properties of the microenvironment within microtissues that makes them a promising next-generation therapeutic approach.

Intramyocardial transplantation and tracking of human mesenchymal stem cells in a novel intra-uterine pre-immune fetal sheep myocardial infarction model: a proof of concept study.

Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70–75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7–9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×105–5×105 human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific β-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow-Cytometry detected the cells within spleen, lungs, kidneys, thymus, bone-marrow and intra-peritoneal lavage, but not within the heart. For the first time we demonstrate the feasibility of intra-uterine, intra-myocardial stem-cell transplantation into the pre-immune fetal-sheep after MI. Utilizing cell-tracking strategies comprising advanced imaging-technologies and in-vitro tracking-tools, this novel model may serve as a unique platform to assess human cell-fate after intra-myocardial transplantation without the necessity of immunosuppressive-therapy.

Cardiac Regenerative Medicine: The Potential of a New Generation of Stem Cells.

Cardiac stem cell therapy holds great potential to prompt myocardial regeneration in patients with ischemic heart disease. The selection of the most suitable cell type is pivotal for its successful application. Various cell types, including crude bone marrow mononuclear cells, skeletal myoblast, and hematopoietic and endothelial progenitors, have already advanced into the clinical arena based on promising results from different experimental and preclinical studies. However, most of these so-called first-generation cell types have failed to fully emulate the promising preclinical data in clinical trials, resulting in heterogeneous outcomes and a critical lack of translation. Therefore, different next-generation cell types are currently under investigation for the treatment of the diseased myocardium. This review article provides an overview of current stem cell therapy concepts, including the application of cardiac stem (CSCs) and progenitor cells (CPCs) and lineage commitment via guided cardiopoiesis from multipotent cells such as mesenchymal stem cells (MSCs) or pluripotent cells such as embryonic and induced pluripotent stem cells. Furthermore, it introduces new strategies combining complementary cell types, such as MSCs and CSCs/CPCs, which can yield synergistic effects to boost cardiac regeneration.

Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types

Acute myocardial infarction and chronic heart failure rank among the major causes of morbidity and mortality worldwide. Except for heart transplantation, current therapy options only treat the symptoms but do not cure the disease. Stem cell-based therapies represent a possible paradigm shift for cardiac repair. However, most of the first-generation approaches displayed heterogeneous clinical outcomes regarding efficacy. Stemming from the desire to closely match the target organ, second-generation cell types were introduced and rapidly moved from bench to bedside. Unfortunately, debates remain around the benefit of stem cell therapy, optimal trial design parameters, and the ideal cell type. Aiming at highlighting controversies, this article provides a critical overview of the translation of first-generation and second-generation cell types. It further emphasizes the importance of understanding the mechanisms of cardiac repair and the lessons learned from first-generation trials, in order to improve cell-based therapies and to potentially finally implement cell-free therapies.

Antibody Phage Display Assisted Identification of Junction Plakoglobin as a Potential Biomarker for Atherosclerosis

To date, no plaque-derived blood biomarker is available to allow diagnosis, prognosis or monitoring of atherosclerotic vascular diseases. In this study, specimens of thrombendarterectomy material from carotid and iliac arteries were incubated in protein-free medium to obtain plaque and control secretomes for subsequent subtractive phage display. The selection of nine plaque secretome-specific antibodies and the analysis of their immunopurified antigens by mass spectrometry led to the identification of 22 proteins. One of them, junction plakoglobin (JUP-81) and its smaller isoforms (referred to as JUP-63, JUP-55 and JUP-30 by molecular weight) were confirmed by immunohistochemistry and immunoblotting with independent antibodies to be present in atherosclerotic plaques and their secretomes, coronary thrombi of patients with acute coronary syndrome (ACS) and macrophages differentiated from peripheral blood monocytes as well as macrophage-like cells differentiated from THP1 cells. Plasma of patients with stable coronary artery disease (CAD) (n = 15) and ACS (n = 11) contained JUP-81 at more than 2- and 14-fold higher median concentrations, respectively, than plasma of CAD-free individuals (n = 13). In conclusion, this proof of principle study identified and verified JUP isoforms as potential plasma biomarkers for atherosclerosis. Clinical validation studies are needed to determine its diagnostic efficacy and clinical utility as a biomarker for diagnosis, prognosis or monitoring of atherosclerotic vascular diseases.