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Stem cell-based therapies are considered to be promising and innovative therapeutic strategies for heart repair. Patient-derived induced pluripotent stem cells (iPSCs) are now available, which combine the advantages of autologous adult stem cells with the unlimited potential of embryonic stem cells for proliferation and differentiation. Intense research has driven dramatic progress in various areas of iPSC technology relevant for clinically applicable iPSC-based cellular therapies. At this point, it is already possible to generate transgene-free autologous iPSCs from small blood samples or hair, to scale up the expansion and differentiation of iPSCs to clinically required dimensions, and to obtain highly enriched cardiomyocyte preparations. On the other hand, critical hurdles such as the targeted specification of distinct cardiomyocyte subpopulations, survival and proper functional integration of cellular transplants after myocardial infarction, and in vitro engineering of prevascularized muscle patches have yet to be overcome. Nevertheless, concepts of cellular cardiomyoplasty seem to have come of age and the first clinical applications of iPSC-based heart repair can be expected within the coming years.

Disease-specific induced pluripotent stem cells (iPSCs) are invaluable tools for studying genetic disorders in a dish. A recent paper by Wang et al. (2014) powerfully combines analysis of human iPSCs with genome editing and tissue engineering, in conjunction with biochemical and physiological assays, to provide insights into Barth-syndrome-associated cardiomyopathy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality., Methods and Results: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition., Conclusion: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

Despite recent major advances including reprogramming and directed cardiac differentiation of human cells, therapeutic application of in vitro engineered myocardial tissue is still not feasible due to the inability to construct functional large vascularized contractile tissue patches based on clinically applicable and fully defined matrix components. Typical matrices with preformed porous 3D structure cannot be applied due to the obvious lack of migratory capacity of cardiomyocytes (CM). We have therefore developed a fully defined in situ hydrogelation system based on alginate (Alg) and hyaluronic acid (HyA), in which their aldehyde and hydrazide-derivatives enable covalent hydrazone cross-linking of polysaccharides in the presence of viable myocytes. By varying degrees of derivatization, concentrations and composition of blends in a modular system, mechanophysical properties of the resulting hydrogels are easily adjustable. The hydrogel allowed for the generation of contractile bioartificial cardiac tissue from CM-enriched neonatal rat heart cells, which resembles native myocardium. A combination of HyA and highly purified human collagen I led to significantly increased active contraction force compared to collagen, only. Therefore, our in situ cross-linking hydrogels represent a valuable toolbox for the fine-tuning of engineered cardiac tissue's mechanical properties and improved functionality, facilitating clinical translation toward therapeutic heart muscle reconstruction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Stem cell-based cardiac tissue engineering is a promising approach for regenerative therapy of the injured heart. At present, the small number of stem cell-derived cardiomyocytes that can be obtained using current culture and enrichment techniques represents one of the key limitations for the development of functional bioartificial cardiac tissue (BCT). We have addressed this problem by construction of a novel bioreactor with functional features of larger systems that enables the generation and in situ monitoring of miniaturized BCTs. BCTs were generated from rat cardiomyocytes to demonstrate advantages and usefulness of the bioreactor. Tissues showed spontaneous, synchronized contractions with cell orientation along the axis of strain. Cyclic stretch induced cardiomyocyte hypertrophy, demonstrated by a shift of myosin heavy chain expression from the alpha to beta isoform, together with elevated levels of atrial natriuretic factor. Stretch led to a moderate increase in systolic force (1.42 ± 0.09 mN vs. 0.96 ± 0.09 mN in controls), with significantly higher forces observed after β-adrenergic stimulation with noradrenalin (2.54 ± 0.11 mN). Combined mechanical and β-adrenergic stimulation had no synergistic effect. This study demonstrates for the first time that mechanical stimulation and direct real-time contraction force measurement can be combined into a single multimodal bioreactor system, including electrical stimulation of excitable tissue, perfusion of the culture chamber, and the possibility of (fluorescence) microscopic assessment during continuous cultivation. Thus, this bioreactor represents a valuable tool for monitoring tissue development and, ultimately, the optimization of stem cell-based tissue replacement strategies in regenerative medicine.

Polymeric materials used in biomedical devices, bioartificial organs, or for the fabrication of tissue engineering scaffolds should completely prevent the activation of the coagulation system and subsequent clot formation. Surface endothelialization is considered an important tool to optimize the blood compatibility of synthetic materials, as a functional endothelial cell layer on an artificial material may help control hemostasis and, therefore, provide a solution to improve the biocompatibility of these materials. Here we report on the endothelialization of poly 4-methyl-1-pentene (PMP) gas exchange membranes using human cord blood-derived late outgrowth endothelial colony forming cells. We achieved complete endothelialization of PMP membranes; and when seeded and cultivated on the membrane, cord blood-derived late outgrowth endothelial colony forming cells maintained both endothelial characteristics and functionality. Endothelialization resulted in significantly lower platelet adhesion and activation compared with unseeded membranes. Of importance, the endothelial layer had no major impact on gas permeability of PMP membranes. This study is a first promising step toward the development of a biofunctionalized surface for the use in gas exchange devices with blood contacting surfaces and a straightforward approach toward a long-term bio-hybrid lung replacement system.

The induction of pluripotency in somatic cells is widely considered as a major breakthrough in regenerative medicine, because this approach provides the basis for individualized stem cell-based therapies. Moreover, with respect to cell transplantation and tissue engineering, expertise from bioengineering to transplantation medicine is now meeting basic research of stem cell biology.In this chapter, we discuss techniques, potential and possible risks of induced pluripotent stem (iPS) cells in the light of needs for patient-derived pluripotent stem cells. To this end, we compare these cells with other sources of pluripotent cells and discuss the first encouraging results of iPS cells in pharmacological research, disease modeling and cell transplantation, providing fascinating perspectives for future developments in biotechnology and regenerative medicine.

Background: The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease- and human patient-specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line., Methods and Results: With the use of a standard embryoid body-based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca(2+) fluctuations with amplitudes of Ca(2+) transients comparable to ES cell cardiomyocytes. Simultaneous Ca(2+) release within clusters of iPS cell-derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta-adrenergic and muscarinic signaling cascade in these cells., Conclusions: iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies. [ABSTRACT FROM AUTHOR]

Background: The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections. Methods and Results: A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5×105) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78×105±0.31×105 in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74×105±0.18×105; p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90×105±0.20×105) and the right (1.07×105±0.17×105) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups. Conclusions: We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs. [ABSTRACT FROM AUTHOR]