Multi-organ platform with tissue-specific niches linked by vascular flow for studies of systemic disease.

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Engineering Adult-like Human Myocardium for Predictive Models of Cardiotoxicity and Disease

Preclinical screening during the development of new drugs is poorly predictive and costly, creating a significant interest from pharmaceutical companies, government agencies, and the public in the development of better preclinical tests. To create more predictive organ models, human derived stem cells can be coupled with biomimetic tissue engineering approaches to create physiologically relevant functional subunits of each tissue/organ within the body. However, existing methods of generating cardiomyocytes (CMs) and cardiac tissues from human induced pluripotent stem cells (hiPSC) derived CMs (hiPS-CMs) are relatively immature and produce tissues that resemble that of a fetal heart at best. This limits their use in therapeutic development and thus, methods to overcome their immature phenotype are of high importance. In pursuit of this goal, this dissertation focuses on the role of biophysical stimuli in driving the functional maturation of hiPSC-CMs to engineer cardiac muscle of high biological fidelity. In an effort to recapitulate the hierarchical structure and functionality of native heart tissue, methods to pattern cells at the nano- and microscale levels were developed and optimized towards the functional assembly of cardiac tissues at the macroscale. To address the challenges currently associated with hiPS-CM immaturity, the decoupled effects of electrical and electromechanical stimulation in driving cardiac maturation were investigated. Subsequently, optimal electromechanical stimulation regimens were established. Daily intervals of high intensity electromechanical training were shown to upregulate cardiac functionality and energetics, and thus, enhance maturation. Combining these methods enabled the development of a custom bioreactor capable of generating larger, more functionally mature hiPS-CM tissues. Mimicking the developmental increases in cardiac beating frequency, exposure of the resulting tissues to a dynamic electromechanical intensity training regimen matured hiPS-CMs beyond levels currently demonstrated within the field. Specifically, the engineered tissues recapitulated many of the molecular, structural, and functional properties of adult human heart muscle, including well developed registers of sarcomeres, networks of T-tubules, calcium homeostasis, and a positive force-frequency relationship. The enhanced functionality of the resulting bio-engineered adult-like myocardium enabled its utility in predicting drug cardiotoxicity and modeling human cardiac disease

Micropatterning of Cells Reveals Chiral Morphogenesis

Invariant left-right (LR) patterning or chirality is critical for embryonic development. The loss or reversal of LR asymmetry is often associated with malformations and disease. Although several theories have been proposed, the exact mechanism of the initiation of the LR symmetry has not yet been fully elucidated. Recently, chirality has been detected within single cells as well as multicellular structures using several in vitro approaches. These studies demonstrated the universality of cell chirality, its dependence on cell phenotype, and the role of physical boundaries. In this review, we discuss the theories for developmental LR asymmetry, compare various in vitro cell chirality model systems, and highlight possible roles of cell chirality in stem cell differentiation. We emphasize that the in vitro cell chirality systems have great promise for helping unveil the nature of chiral morphogenesis in development

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype

Author ManuscriptThe application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR, and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); NSF grant 16478 (G.V.-N.); the University of Minho MD/PhD program (D.T.); a Japan Society for the Promotion of Science fellowship (K.M.); and the Columbia University Stem Cell Initiative (L.S., M.Y.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.The authors acknowledge funding support from the National Institutes of Health of the USA (NIBIB and NCATS grant EB17103 (G.V.-N.); NIBIB, NCATS, NIAMS, NIDCR and NIEHS grant EB025765 (G.V.-N.); NHLBI grants HL076485 (G.V.-N.) and HL138486 (M.Y.); Columbia University MD/PhD program (S.P.M., T.C.); University of Minho MD/PhD program (D.T.); Japan Society for the Promotion of Science fellowship (K.M.); and Columbia University Stem Cell Initiative (D.S., L.S., M.Y.). We thank S. Duncan and B. Conklin for providing human iPSCs, M.B. Bouchard for assistance with image and video analysis, and L. Cohen-Gould for transmission electron microscopy services.info:eu-repo/semantics/publishedVersio

Electromechanical stimulation advances maturation of human cardiac tissues grown from pluripotent stem cells

Data used to generate the figures in the manuscript Electromechanical stimulation advances maturation of human cardiac tissues grown from pluripotent stem cells

Hierarchically ordered nanopatterns for spatial control of biomolecules.

The development and study of a benchtop, high-throughput, and inexpensive fabrication strategy to obtain hierarchical patterns of biomolecules with sub-50 nm resolution is presented. A diblock copolymer of polystyrene-b-poly(ethylene oxide), PS-b-PEO, is synthesized with biotin capping the PEO block and 4-bromostyrene copolymerized within the polystyrene block at 5 wt %. These two handles allow thin films of the block copolymer to be postfunctionalized with biotinylated biomolecules of interest and to obtain micropatterns of nanoscale-ordered films via photolithography. The design of this single polymer further allows access to two distinct superficial nanopatterns (lines and dots), where the PEO cylinders are oriented parallel or perpendicular to the substrate. Moreover, we present a strategy to obtain hierarchical mixed morphologies: a thin-film coating of cylinders both parallel and perpendicular to the substrate can be obtained by tuning the solvent annealing and irradiation conditions

Hierarchically Ordered Nanopatterns for Spatial Control of Biomolecules

The development and study of a benchtop, high-throughput, and inexpensive fabrication strategy to obtain hierarchical patterns of biomolecules with sub-50 nm resolution is presented. A diblock copolymer of polystyrene-<i>b</i>-poly(ethylene oxide), PS-<i>b</i>-PEO, is synthesized with biotin capping the PEO block and 4-bromostyrene copolymerized within the polystyrene block at 5 wt %. These two handles allow thin films of the block copolymer to be postfunctionalized with biotinylated biomolecules of interest and to obtain micropatterns of nanoscale-ordered films <i>via</i> photolithography. The design of this single polymer further allows access to two distinct superficial nanopatterns (lines and dots), where the PEO cylinders are oriented parallel or perpendicular to the substrate. Moreover, we present a strategy to obtain hierarchical mixed morphologies: a thin-film coating of cylinders both parallel and perpendicular to the substrate can be obtained by tuning the solvent annealing and irradiation conditions

A guide to the organ-on-a-chip

Organs-on-chips (OoCs) are systems containing engineered or natural miniature tissues grown inside microfluidic chips. To better mimic human physiology, the chips are designed to control cell microenvironments and maintain tissue-specific functions. Combining advances in tissue engineering and microfabrication, OoCs have gained interest as a next-generation experimental platform to investigate human pathophysiology and the effect of therapeutics in the body. There are as many examples of OoCs as there are applications, making it difficult for new researchers to understand what makes one OoC more suited to an application than another. This Primer is intended to give an introduction to the aspects of OoC that need to be considered when developing an application-specific OoC. The Primer covers guiding principles and considerations to design, fabricate and operate an OoC, as well as subsequent assaying techniques to extract biological information from OoC devices. Alongside this is a discussion of current and future applications of OoC technology, to inform design and operational decisions during the implementation of OoC systems