Until recently, cells were thought to be integral and discrete components of tissues, and their state was determined by cell differentiation. However, under some conditions, stem cells or their progeny can fuse with cells of other types, mixing cytoplasmic and even genetic material of different (heterotypic) origins. The fusion of heterotypic cells could be of central importance for development, repair of tissues and the pathogenesis of disease.

Rationale Conventional three-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective Here, we used multiphoton-excited, 3-dimensional printing (MPE-3DP) to generate a native-like, extracellular matrix (ECM) scaffold with submicron resolution, and then seeded the scaffold with cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs) that had been differentiated from human induced-pluripotent stem cells (iPSCs) to generate a human, iPSC-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction (MI). Methods and Results The scaffold was seeded with ~50,000 human, iPSC-derived CMs, SMCs, and ECs (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced MI, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions Thus, the novel MPE-3DP technique produces ECM-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes—a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. Objective: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. Methods and Results: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell–laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. Conclusions: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or “big questions” were articulated that, if addressed, could substantially advance the current state-of-the-art in modeling heart disease and realizing heart repair.

Although cancer cell fusion has been suggested as a mechanism of cancer metastasis, the underlying mechanisms defining this process are poorly understood. In a recent study, apoptotic cells were newly identified as a type of cue that induces signaling via phosphatidylserine receptors to promote fusion of myoblasts. The microenvironment of breast tumors is often hypoxic, and because apoptosis is greatly increased in hypoxic conditions, we decided to investigate whether the mechanism of breast cancer cell fusion with mesenchymal stem/multipotent stromal cells (MSCs) involves apoptosis. We used a powerful tool for identification and tracking of hybrids based on bimolecular fluorescence complementation (BiFC) and found that breast cancer cells fused spontaneously with MSCs. This fusion was significantly enhanced with hypoxia and signaling associated with apoptotic cells, especially between nonmetastatic breast cancer cells and MSCs. In addition, the hybrids showed a significantly higher migratory capacity than did the parent cells. Taken together, these findings describe a mechanism by which hypoxiainduced apoptosis stimulates fusion between MSCs and breast tumor cells resulting in hybrids with an enhanced migratory capacity that may enable their dissemination to distant sites or metastases. In the long run, this study may provide new strategies for developing novel drugs for preventing cancer metastasis.-Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis. FASEB J. 29, 4036-4045 (2015). www.fasebj.org

The influence of specific serum-borne biomolecules (e.g. heparin) on growth factor-dependent cell behavior is often difficult to elucidate in traditional cell culture due to the random, non-specific nature of biomolecule adsorption from serum. We hypothesized that chemically well-defined cell culture substrates could be used to study the influence of sequestered heparin on human mesenchymal stem cell (hMSC) behavior. Specifically, we used bio-inert self-assembled monolayers (SAMs) chemically modified with a bioinspired heparin-binding peptide (termed “HEPpep”) and an integrin-binding peptide (RGDSP) as stem cell culture substrates. Our results demonstrate that purified heparin binds to HEPpep SAMs in a dose-dependent manner, and serum-borne heparin binds specifically and in a dose-dependent manner to HEPpep SAMs. These heparin-sequestering SAMs enhance hMSC proliferation by amplifying endogenous fibroblast growth factor (FGF) signaling, and enhance hMSC osteogenic differentiation by amplifying endogenous bone morphogenetic protein (BMP) signaling. The effects of heparin-sequestering are similar to the effects of supraphysiologic concentrations of recombinant FGF-2. hMSC phenotype is maintained over multiple population doublings on heparin-sequestering substrates in growth medium, while hMSC osteogenic differentiation is enhanced in a bone morphogenetic protein-dependent manner on the same substrates during culture in osteogenic induction medium. Together, these observations demonstrate that the influence of the substrate on stem cell phenotype is sensitive to the culture medium formulation. Our results also demonstrate that enhanced hMSC proliferation can be spatially localized by patterning the location of HEPpep on the substrate. Importantly, the use of chemically well-defined SAMs in this study eliminated the confounding factor of random, non-specific biomolecule adsorption, and identified serum-borne heparin as a key mediator of hMSC response to endogenous growth factors.

Solid organ fabrication is an ultimate goal of Regenerative Medicine. Since the introduction of Tissue Engineering in 1993, functional biomaterials, stem cells, tunable microenvironments, and high-resolution imaging technologies have significantly advanced efforts to regenerate in vitro culture or tissue platforms. Relatively simple flat or tubular organs are already in (pre)clinical trials and a few commercial products are in market. The road to more complex, high demand, solid organs including heart, kidney and lung will require substantive technical advancement. Here, we consider two emerging technologies for solid organ fabrication. One is decellularization of cadaveric organs followed by repopulation with terminally differentiated or progenitor cells. The other is 3D bioprinting to deposit cell-laden bio-inks to attain complex tissue architecture. We reviewed the development and evolution of the two technologies and evaluated relative strengths needed to produce solid organs, with special emphasis on the heart and other tissues of the cardiovascular system.

We report Fourier transform infrared spectro-microtomography, a nondestructive three-dimensional imaging approach that reveals the distribution of distinctive chemical compositions throughout an intact biological or materials sample. The method combines mid-infrared absorption contrast with computed tomographic data acquisition and reconstruction to enhance chemical and morphological localization by determining a complete infrared spectrum for every voxel (millions of spectra determined per sample).