Cellular population dynamics shape the route to human pluripotency.

Human cellular reprogramming to induced pluripotency is still an inefficient process, which has hindered studying the role of critical intermediate stages. Here we take advantage of high efficiency reprogramming in microfluidics and temporal multi-omics to identify and resolve distinct sub-populations and their interactions. We perform secretome analysis and single-cell transcriptomics to show functional extrinsic pathways of protein communication between reprogramming sub-populations and the re-shaping of a permissive extracellular environment. We pinpoint the HGF/MET/STAT3 axis as a potent enhancer of reprogramming, which acts via HGF accumulation within the confined system of microfluidics, and in conventional dishes needs to be supplied exogenously to enhance efficiency. Our data suggest that human cellular reprogramming is a transcription factor-driven process that it is deeply dependent on extracellular context and cell population determinants.

3D ECM-rich environment sustains the identity of naive human iPSCs.

The establishment of in vitro naive human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways have been characterized during maintenance of human naive pluripotency. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining microfluidic cultures with transcriptional, proteomic, and secretome analyses, we found that naive, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naive hiPSCs and also allows direct and timely developmental morphogenesis simply by modulating the signaling environment. Our study opens new perspectives for future applications of naive hiPSCs to study critical stages of human development in 3D starting from a single cell.

Extracellular phosphoprotein regulation is affected by culture system scale-down.

BACKGROUND: Phosphorylated proteins are known to be present in multiple body fluids in normal conditions, and abnormally accumulated under some pathological conditions. The biological significance of their role in the extracellular space has started being elucidated only recently, for example in bone mineralization, neural development, and coagulation. Here, we address some criticalities of conventional culture systems for the study of the extracellular regulation of phosphorylation. METHODS: We make use of microfluidics to scale-down the culture volume to a size comparable to the interstitial spaces occurring in vivo. The phosphoprotein content of conditioned media was analyzed by a colorimetric assay that detects global phosphorylation. RESULTS: We found that miniaturization of the culture system increases phosphoprotein accumulation. Moreover, we demonstrated that in conventional culture systems dilution affects the extent of the phosphorylation reactions occurring within the extracellular space. On the other hand, in microfluidics the phosphorylation status was not affected by addition of adenosine triphosphate (ATP) and FAM20C Golgi Associated Secretory Pathway Kinase (FAM20C) ectokinase, as if their concentration was already not limiting for the phosphorylation reaction to occur. CONCLUSIONS: The volume of the extracellular environment plays a role in the process of extracellular phosphorylation due to its effect on the concentration of substrates, enzymes and co-factors. GENERAL SIGNIFICANCE: Thus, the biological role of extracellular phosphoregulation may be better appreciated within a microfluidic culture system.

Micropillar-based phenotypic screening platform uncovers involvement of HDAC2 in nuclear deformability.

Nuclear deformation is an essential phenomenon allowing cell migration and can be observed in association with pathological conditions such as laminopathies, neurodegenerative disorders and diabetes. Abnormal nuclear morphologies are a hallmark of cancer progression and nuclear deformability is a necessary feature for metastatic progression. Nevertheless, the cellular processes and the key molecular components controlling nuclear shape are poorly understood, in part due to a limited availability of assays that allow high-throughput screening of nuclear morphology-phenotypes. In this study, we explore the application of micropillared substrates as the basis for a phenotypic screening platform aimed at identifying novel determinants of nuclear morphology. We designed PDMS substrates to maximize simplicity in image acquisition and analyses, and in a small-scale screening of inhibitors targeting chromatin-modifying enzymes, we identify histone deacetylation as cellular process involved in nuclear deformation. With increasingly specific targeting approaches, we identify HDAC2 as a novel player in controlling nuclear morphology through gene transcription repression. This study shows the effectiveness of micropillar-based substrates to act as phenotypic drug screening platforms and opens a new avenue in the identification of genes involved in determining the nuclear shape.

Nuclear Morphological Remodeling in Human Granulocytes Is Linked to Prenylation Independently from Cytoskeleton.

Nuclear shape modulates cell behavior and function, while aberrant nuclear morphologies correlate with pathological phenotype severity. Nevertheless, functions of specific nuclear morphological features and underlying molecular mechanisms remain poorly understood. Here, we investigate a nucleus-intrinsic mechanism driving nuclear lobulation and segmentation concurrent with granulocyte specification, independently from extracellular forces and cytosolic cytoskeleton contributions. Transcriptomic regulation of cholesterol biosynthesis is equally concurrent with nuclear remodeling. Its putative role as a regulatory element is supported by morphological aberrations observed upon pharmacological impairment of several enzymatic steps of the pathway, most prominently the sterol ∆14-reductase activity of laminB-receptor and protein prenylation. Thus, we support the hypothesis of a nuclear-intrinsic mechanism for nuclear shape control with the putative involvement of the recently discovered GGTase III complex. Such process could be independent from or complementary to the better studied cytoskeleton-based nuclear remodeling essential for cell migration in both physiological and pathological contexts such as immune system function and cancer metastasis.

Beyond Family: Modeling Non-hereditary Heart Diseases With Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Non-genetic cardiac pathologies develop as an aftermath of extracellular stress-conditions. Nevertheless, the response to pathological stimuli depends deeply on intracellular factors such as physiological state and complex genetic backgrounds. Without a thorough characterization of their in vitro phenotype, modeling of maladaptive hypertrophy, ischemia and reperfusion injury or diabetes in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has been more challenging than hereditary diseases with defined molecular causes. In past years, greater insights into hPSC-CM in vitro physiology and advancements in technological solutions and culture protocols have generated cell types displaying stress-responsive phenotypes reminiscent of in vivo pathological events, unlocking their application as a reductionist model of human cardiomyocytes, if not the adult human myocardium. Here, we provide an overview of the available literature of pathology models for cardiac non-genetic conditions employing healthy (or asymptomatic) hPSC-CMs. In terms of numbers of published articles, these models are significantly lagging behind monogenic diseases, which misrepresents the incidence of heart disease causes in the human population.

Photocrosslinked hydrogels from coumarin derivatives of hyaluronic acid for tissue engineering applications.

Hydrogels are an increasingly attractive choice in the fields of regenerative medicine, wound care and tissue engineering as important forms of bio-scaffolds. For many clinical needs, injectable in situ crosslinkable hydrogels are strongly preferred, due to treatment effectiveness and ease of use. In this study, hyaluronic acid (HA), containing side-arms linked to photo-active coumarin moieties, was used for the preparation of wall-to-wall hydrogels. This photocrosslinkable HA, hereafter called HA-TEG-coumarin, produces colourless aqueous solutions that solidify upon near-UV irradiation (at a specific wavelength of 365 nm) via a clean [2 + 2] photocycloaddition reaction, without by-products formation. The crosslinking event, a robust and non-cytotoxic process, does not require catalysts or radical initiators: in the field of hyaluronan photocrosslinking, this innovative feature is significant to ensure the whole biocompatibility and to avoid collateral reactions. Mechanical and rheological tests showed that hyaluronan derivatives became hydrogels after 3-5 min of irradiation, with average values for bulk and surface elastic moduli of about 32 kPa and 193 kPa, respectively. Fluorescence recovery after photobleaching (FRAP) assay showed that the hydrogels are porous and allow a good permeation for nutrients and growth factors. Cell metabolism and proliferation assays revealed that hydrogel-encapsulated fibroblasts maintained their viability and that HA-TEG-coumarin sustained the proliferation of non-adherent myoblasts. For all of these reasons and thanks to a safe free-radical approach, this novel hyaluronan coumarin derivative could be a good candidate for tissue engineering and regenerative medicine applications.

Transcriptomic Characterization of a Human In Vitro Model of Arrhythmogenic Cardiomyopathy Under Topological and Mechanical Stimuli.

Cell junctions play an important role in coordinating intercellular communication and intracellular ultrastructures, with desmosomes representing the mechanical component of such intercellular connections. Mutations to desmosomal component proteins compromise both inter- and intracellular signalling and correlate with severe diseases like arrhythmogenic cardiomyopathy (AC), with pathological phenotypes in tissues subjected to intense mechanical stimuli (skin and heart). Here, we explore the consequences of dysfunctional desmosomes in one line of induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) derived from an AC patient with a homozygous pathogenic mutation in desmosomal component protein plakophilin-2 (PKP2). We specifically aim at investigating the response to mechanical stress in an AC-pathological setting. To this aim, we aligned hiPS-CMs on stretchable patterned substrates to mimic the cardiac functional syncytium and compared transcriptomic profiles of PKP2-mutated hiPS-CMs and healthy controls. AC-CMs display altered transcription towards a pro-fibrotic gene expression program, and concurrent dysregulation of gene sets closely associated with cell-to-cell connections. By integrating the culture substrate with a macroscopic stretching setup able to accurately apply cyclic uniaxial elongation, we show how response to mechanical loads in AC-CMs deviates from the canonical mechanical-stress response observed in healthy-CMs.

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO2 environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism in vitro.

Substrate and mechanotransduction influence SERCA2a localization in human pluripotent stem cell-derived cardiomyocytes affecting functional performance.

Physical cues are major determinants of cellular phenotype and evoke physiological and pathological responses on cell structure and function. Cellular models aim to recapitulate basic functional features of their in vivo counterparts or tissues in order to be of use in in vitro disease modeling or drug screening and testing. Understanding how culture systems affect in vitro development of human pluripotent stem cell (hPSC)-derivatives allows optimization of cellular human models and gives insight in the processes involved in their structural organization and function. In this work, we show involvement of the mechanotransduction pathway RhoA/ROCK in the structural reorganization of hPSC-derived cardiomyocytes after adhesion plating. These structural changes have a major impact on the intracellular localization of SERCA2 pumps and concurrent improvement in calcium cycling. The process is triggered by cell interaction with the culture substrate, which mechanical cues drive sarcomeric alignment and SERCA2a spreading and relocalization from a perinuclear to a whole-cell distribution. This structural reorganization is mediated by the mechanical properties of the substrate, as shown by the process failure in hPSC-CMs cultured on soft 4kPa hydrogels as opposed to physiologically stiff 16kPa hydrogels and glass. Finally, pharmacological inhibition of Rho-associated protein kinase (ROCK) by different compounds identifies this specific signaling pathway as a major player in SERCA2 localization and the associated improvement in hPSC-CMs calcium handling ability in vitro.

Analysis of Calcium Transients and Uniaxial Contraction Force in Single Human Embryonic Stem Cell-Derived Cardiomyocytes on Microstructured Elastic Substrate with Spatially Controlled Surface Chemistries.

The mechanical activity of cardiomyocytes is the result of a process called excitation-contraction coupling (ECC). A membrane depolarization wave induces a transient cytosolic calcium concentration increase that triggers activation of calcium-sensitive contractile proteins, leading to cell contraction and force generation. An experimental setup capable of acquiring simultaneously all ECC features would have an enormous impact on cardiac drug development and disease study. In this work, we develop a microengineered elastomeric substrate with tailor-made surface chemistry to measure simultaneously the uniaxial contraction force and the calcium transients generated by single human cardiomyocytes in vitro. Microreplication followed by photocuring is used to generate an array consisting of elastomeric micropillars. A second photochemical process is employed to spatially control the surface chemistry of the elastomeric pillar. As result, human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be confined in rectangular cell-adhesive areas, which induce cell elongation and promote suspended cell anchoring between two adjacent micropillars. In this end-to-end conformation, confocal fluorescence microscopy allows simultaneous detection of calcium transients and micropillar deflection induced by a single-cell uniaxial contraction force. Computational finite elements modeling (FEM) and 3D reconstruction of the cell-pillar interface allow force quantification. The platform is used to follow calcium dynamics and contraction force evolution in hESC-CMs cultures over the course of several weeks. Our results show how a biomaterial-based platform can be a versatile tool for in vitro assaying of cardiac functional properties of single-cell human cardiomyocytes, with applications in both in vitro developmental studies and drug screening on cardiac cultures.

Functional differentiation of human pluripotent stem cells on a chip.

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD)-associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient-derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.