Impact of Poly(dimethylsiloxane) Surface Modification with Conventional and Amino Acid-Conjugated Self-Assembled Monolayers on the Differentiation of Induced Pluripotent Stem Cells into Cardiomyocytes.

Cardiomyocytes, differentiated from induced pluripotent stem cells (iPSCs), have the potential to produce patient- and disease-specific pharmacological and toxicological platforms, in addition to their cardiac cell therapy applications. However, the lack of both a robust and a simple procedure for scalable cell substrate production is one of the major limitations in this area. Mimicking the natural healthy myocardium extracellular matrix (ECM) properties by altering the cell substrate properties, such as stiffness and chemical/biochemical composition, can significantly affect cell substrate interfacial characteristics and potentially influence cellular behavior and differentiation of iPSCs to cardiomyocytes. Here, we propose a systematic and biomimetic approach, based on the preparation of poly(dimethylsiloxane) (PDMS) substrates having the similar stiffness as healthy heart tissue and a well-defined surface chemistry obtained by conventional [(3-aminopropyl)triethoxysilane (APTES) and octadecyltrimethoxysilane (OTS)] and amino acid (histidine and leucine)-conjugated self-assembled monolayers (SAMs). Among a wide range of different concentrations, the 50:1 prepolymer cross-linker ratio of PDMS allowed adaptation of the myocardium stiffness with a Young's modulus of 23.79 ± 0.61 kPa. Compared with conventional SAM modification, amino acid-conjugated SAMs greatly improved iPSC adhesion, viability, and cardiac marker expression by increasing surface biomimetic properties, whereas all SAMs enhanced cell behavior, with respect to native PDMS. Furthermore, leucine-conjugated SAM modification provided the best environment for cardiac differentiation of iPSCs. This optimized approach can be easily adapted for cardiac differentiation of iPSCs in vitro, rendering a very promising tool for microfluidics, drug screening, and organ-on-chip platforms.

In Vitro Grown Micro-Tissues for Cardiac Cell Replacement Therapy in Vivo.

BACKGROUND/AIMS: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice. METHODS: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections. RESULTS: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging. CONCLUSION: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.

Cardiomyocytes facing fibrotic conditions re-express extracellular matrix transcripts.

Pathophysiological conditions, such as myocardial infarction and mechanical overload affect the mammalian heart integrity, leading to a stiffened fibrotic tissue. With respect to the pathophysiology of cardiac fibrosis but also in the limelight of upcoming approaches of cardiac cell therapy it is of interest to decipher the interaction of cardiomyocytes with fibrotic matrix. Therefore, we designed a hydrogel-based model to engineer fibrotic tissue in vitro as an approach to predict the behavior of cardiomyocytes facing increased matrix rigidity. Here, we generated pure induced pluripotent stem cell-derived cardiomyocytes and cultured them on engineered polyacrylamide hydrogels matching the elasticities of healthy as well as fibrotic cardiac tissue. Only in cardiomyocytes cultured on matrices with fibrotic-like elasticity, transcriptional profiling revealed a substantial up-regulation of a whole panel of cardiac fibrosis-associated transcripts, including collagen I and III, decorin, lumican, and periostin. In addition, matrix metalloproteinases and their inhibitors, known to be essential in cardiac remodeling, were found to be elevated as well as insulin-like growth factor 2. Control experiments with primary cardiac fibroblasts were analyzed and did not show comparable behavior. In conclusion, we do not only present a snapshot on the transcriptomic fingerprint alterations in cardiomyocytes under pathological conditions but also provide a new reproducible approach to study the effects of fibrotic environments to various cell types. STATEMENT OF SIGNIFICANCE: The ageing population in many western countries is faced with an increasing burden of ageing-related diseases such as heart failure which is associated with cardiac fibrosis. A deeper understanding of the interaction of organotypic cells with altered extracellular matrix mechanical properties is of pivotal importance to understand the underlying mechanisms. Here, we present a strategy to combine hydrogel matrices with induced pluripotent stem cell derived cardiomyocytes to study the effect of matrix stiffening on these cells. Our findings suggest an active role of matrix stiffening on cardiomyocyte function and heart failure progression.

The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates.