Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical-stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride coated titanium, titanium), length of stimulus (1 s and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s, and 4-days old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H2O2. Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of Troponin T, and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.

High throughput experiments can be used to spatially and temporally investigate the many factors that regulate cell differentiation. We have developed a micro-bioreactor array (MBA) that is fabricated using soft lithography and contains twelve independent micro-bioreactors perfused with culture medium. The MBA enables cultivation of cells that are either attached to substrates or encapsulated in hydrogels, at variable levels of hydrodynamic shear, and with automated image analysis of the expression of cell differentiation markers. The flow and mass transport in the MBA were characterized by computational fluid dynamic (CFD) modeling. The representative MBA configurations were validated using the C2C12 cell line, primary rat cardiac myocytes and human embryonic stem cells (hESCs) (lines H09 and H13). To illustrate the utility of the MBA for controlled studies of hESCs, we established correlations between the expression of smooth muscle actin and cell density for three different flow configurations.

The success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to engineer myogenic cells and biocompatible scaffolds. Satellite cells (SCs), freshly isolated or transplanted within their niche, are presently considered the best source for muscle regeneration. Here, we designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo-cross-linkable hyaluronan-based hydrogel, hyaluronic acid-photoinitiator (HA-PI) complex. Partially ablated tibialis anterior (TA) of C57BL/6J mice engrafted with freshly isolated satellite cells embedded in hydrogel showed a major improvement in muscle structure and number of new myofibers, compared to muscles receiving hydrogel + MPCs or hydrogel alone. Notably, SCs embedded in HA-PI also promoted functional recovery, as assessed by contractile force measurements. Tissue reconstruction was associated with the formation of both neural and vascular networks and the reconstitution of a functional SC niche. This innovative approach could overcome previous limitations in skeletal muscle tissue engineering.

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

We discuss the utilization of microbioreactor arrays for controlling cellular environments in studies of factors that regulate the differentiation of human embryonic stem cells. To this end, we have designed a simple and practical system that couples a microfluidic platform with an array of microbioreactors, and has the size of a microscope slide [1]. The system allows quantitative studies of cells cultured in monolayers or encapsulated in three-dimensional hydrogels. We review the operating requirements for studies of human embryonic stem cells (hESCs) under steady-state and dynamic conditions, and the related control of the mass transport and hydrodynamic shear. We describe the design and fabrication of the individual bioreactor components, and the criteria for selecting the bioreactor configuration and operating parameters, based on the analysis of the characteristic times and scales of reaction, convection and diffusion. To illustrate the utility of the bioreactor, we present a "case study" of hESC cultivation with detailed experimental methods and representative biological readouts.

Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could be suitable for the production of medical devices that can be beneficial in the field of articular cartilage engineering, thus improving the efficacy and durability of the current therapeutic options.

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SummaryHeart disease is a leading cause of death in western society. Despite the success of heart transplantation, a chronic shortage of donor organs, along with the associated immunological complications of this approach, demands that alternative treatments be found. One such option is to repair, rather than replace, the heart with engineered cardiac tissue. Multiple studies have shown that to attain functional tissue, assembly signaling cues must be recapitulated in vitro. In their native environment, cardiomyocytes are directed to beat in synchrony by propagation of pacing current through the tissue. Recently, we have shown that electrical stimulation directs neonatal cardiomyocytes to assemble into native-like tissue in vitro. This chapter provides detailed methods we have employed in taking this "biomimetic" approach. After an initial discussion on how electric field stimulation can influence cell behavior, we examine the practical aspects of cardiac tissue engineering with electrical stimulation, such as electrode selection and cell seeding protocols, and conclude with what we feel are the remaining challenges to be overcome.