The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction

Rationale: Pluripotent stem cells (PSCs) are being investigated as a cell source for regenerative medicine since they provide an infinitive pool of cells that are able to differentiate towards every cell type of the body. One possible therapeutic application involves the use of these cells to treat myocardial infarction (MI), a condition where billions of cardiomyocytes (CMs) are lost. Although several protocols have been developed to differentiate PSCs towards CMs, none of these provide a completely pure population, thereby still posing a risk for neoplastic teratoma formation. Therefore, we developed a strategy to (i) monitor cell behavior noninvasively via site-specific integration of firefly luciferase (Fluc) and the human positron emission tomography (PET) imaging reporter genes, sodium iodide symporter (hNIS) and somatostatin receptor type 2 (hSSTr2), and (ii) perform hSSTr2-mediated suicide gene therapy via the clinically used radiopharmacon 177Lu-DOTATATE. Methods: Human embryonic stem cells (ESCs) were gene-edited via zinc finger nucleases to express Fluc and either hNIS or hSSTr2 in the safe harbor locus, adeno-associated virus integration site 1. Firstly, these cells were exposed to 4.8 MBq 177Lu-DOTATATE in vitro and cell survival was monitored via bioluminescence imaging (BLI). Afterwards, hNIS+ and hSSTr2+ ESCs were transplanted subcutaneously and teratomas were allowed to form. At day 59, baseline 124I and 68Ga-DOTATATE PET and BLI scans were performed. The day after, animals received either saline or 55 MBq 177Lu-DOTATATE. Weekly BLI scans were performed, accompanied by 124I and 68Ga-DOTATATE PET scans at days 87 and 88, respectively. Finally, hSSTr2+ ESCs were differentiated towards CMs and transplanted intramyocardially in the border zone of an infarct that was induced by left anterior descending coronary artery ligation. After transplantation, the animals were monitored via BLI and PET, while global cardiac function was evaluated using cardiac magnetic resonance imaging. Results: Teratoma growth of both hNIS+ and hSSTr2+ ESCs could be followed noninvasively over time by both PET and BLI. After 177Lu-DOTATATE administration, successful cell killing of the hSSTr2+ ESCs was achieved both in vitro and in vivo, indicated by reductions in total tracer lesion uptake, BLI signal and teratoma volume. As undifferentiated hSSTr2+ ESCs are not therapeutically relevant, they were differentiated towards CMs and injected in immune-deficient mice with a MI. Long-term cell survival could be monitored without uncontrolled cell proliferation. However, no improvement in the left ventricular ejection fraction was observed.Conclusion: We developed isogenic hSSTr2-expressing ESCs that allow noninvasive cell monitoring in the context of PSC-derived regenerative therapy. Furthermore, we are the first to use the hSSTr2 not only as an imaging reporter gene, but also as a suicide mechanism for radionuclide therapy in the setting of PSC-derived cell treatment

Stem cell therapy in muscle degeneration

© Springer Nature Singapore Pte Ltd. 2017. Skeletal muscle is one of the largest tissues in humans, reaching around 38% of the body weight for men and 30% for women (Janssen I, et al, J Appl Physiol 89(1):81-88, 1985/2000). Muscle tissue finely regulates essential body movements, including respiration, equilibrium maintenance and ambulation. In addition, it controls body temperature and energy balance and restores blood glucose levels. Genetic or acquired alterations in these enzymes can lead to metabolic syndromes (McArdle’s syndrome (Vorgerd M, Neurotherapeutics 5(4):579-582 2008) or diabetes (DeFronzo RA, Tripathy D, Diabetes Care 32(Suppl 2):S157- S163 2009). Muscle tissue is originated during embryo development, from the fusion of mesoderm progenitors. During the neonatal and early juvenile period, even if the myofibre number remains constant, new postnatal multipotent cells, called satellite cells (SCs), can fuse and increase the number of nuclei per fibre (Biressi S, et al, Dev Biol 379(2):195-207, 2013). Adult skeletal muscle is organised in long fibres generated from the fusion of single cells into a unique syncytium able to collect thousands of nuclei and of myofibrils. Each myofibril contains multiple sarcomeres, formed by actin and myosin filaments to generate contraction and develop force. High heterogeneity is present among single fibres, including slow-contracting fatigue-resisting type I fibres (also known as slow-twitch, tonic or simply slow fibres) and fast-contracting fatigue-unresisting type II fibres (fasttwitch, phasic or fast fibres). To exert a good performance, the expression of specific contractile proteins and metabolic enzymes is necessary together with a regular connection to a single motor neuron and an efficient vascularisation. Myotendinous junctions connect myofibres from one side and bones in the skeleton on the other side, allowing transmission of contractions to other muscles. Daily wear and tear or excessive load and damage of the fibre integrity activate SCs that undergo fusion. The highly orchestrated process has been thoroughly studied. Nevertheless, understanding the role of local and circulating cell types in adult myogenesis still remains a matter of discussion (Relaix F, Zammit PS, Development 139(16):2845- 2856, 2012). In this chapter after introducing the biological events occurring in acute and chronic skeletal muscle damages, cell and gene therapy approaches are considered as novel tools to preserve muscle function.status: publishe

Genauigkeitskenngrößen von Industrierobotern gemäß ISO 9238 automatisch bestimmen

Die Festlegung des Standards ISO 9283 "Industrial Manipulating Robots - Performance Criteria and Related Testing Methods" ist ein wichtiger Schritt in Richtung der Vereinheitlichung von Genauigkeitsangaben für Industrieroboter. In diesem Aufsatz wird ein System zur vollautomatisierten Vermessung und Ermittlung von Genauigkeitskenngrößen gemäß diesem Standard vorgestellt. Es handelt sich um ein flexibles Softwaresystem , das mit unterschiedlichen Meßsystemen und verschiedenen Robotersteuerungen gekoppelt werden kann, so daß eine individuelle Anpassung an verschiedene Anwendungsanforderungen möglich ist. Es wird ein Meßplatz beschrieben, mit dem die Autoren praktische Erfahrungen mit dem ISO-Standard gesammelt haben. Es existiert eine Reihe offener Fragestellungen bei der Umsetzung des Standards, für die Lösungsansätze angeboten werden. Genauere Analysen von Meßergebnissen gemäß ISO 9283 weisen auf Mängel innerhalb des Standards hin. Eine Diskussion der Ergebnisse, bei der grundsätzliche Probleme behandelt und Änderungsvorschläge gemacht werden, soll gleichzeitig als Diskussionsgrundlage für die 1993/94 zu erwartende Revision des Standards ISO 9283 dienen

Fate choice of post-natal mesoderm progenitors: skeletal versus cardiac muscle plasticity.

Regenerative medicine for skeletal and cardiac muscles still constitutes a fascinating and ambitious frontier. In this perspective, understanding the possibilities of intrinsic cell plasticity, present in post-natal muscles, is vital to define and improve novel therapeutic strategies for acute and chronic diseases. In addition, many somatic stem cells are now crossing the boundaries of basic/translational research to enter the first clinical trials. However, it is still an open question whether a lineage switch between skeletal and cardiac adult myogenesis is possible. Therefore, this review focuses on resident somatic stem cells of post-natal skeletal and cardiac muscles and their plastic potential toward the two lineages. Furthermore, examples of myogenic lineage switch in adult stem cells are also reported and discussed

The ad hoc chemical design of random PBS-based copolymers influences the activation of cardiac differentiation while altering the HYPPO pathway target genes in hiPSCs

Cardiac tissue engineering is a cutting-edge technology aiming to replace irreversibly damaged cardiac tissue and restore contractile functionality. However, cardiac tissue engineering porous and perfusable scaffolds to enable oxygen supply in vitro and eventually promote angiogenesis in vivo are still desirable. Two fully-aliphatic random copolymers of poly(butylene succinate) (PBS), poly(butylene succinate/Pripol), P(BSBPripol), and poly(butylene/neopentyl glycol succinate), P(BSNS), containing two different subunits, neopentyl glycol and Pripol 1009, were successfully synthesized and then electrospun in tridimentional fibrous mats. The copolymers show different thermal and mechanical behaviours as result of their chemical structure. In particular, copolymerization led to a reduction in crystallinity and consequently PBS stiffness, reaching values of elastic modulus very close to those of soft tissues. Then, to check the biological suitability, human induced Pluripotent Stem Cells (hiPSCs) were directly seeded on both PBS-based copolymeric scaffolds. The results confirmed the ability of both the scaffolds to sustain cell viability and to maintain their stemness during cell expansion. Furthermore, gene expression and immunofluorescence analysis showed that P(BSBPripol) scaffold promoted an upregulation of the early cardiac progenitor and later-stage markers with a simultaneously upregulation of HYPPO pathway gene expression, crucial for mechanosensing of cardiac progenitor cells. These results suggest that the correct ad-hoc chemical design and, in turn, the mechanical properties of the matrix, such as substrate stiffness, together with surface porosity, play a critical role in regulating the behaviour of cardiac progenitors, which ultimately offers valuable insights into the development of novel bio-inspired scaffolds for cardiac tissue regeneration