Generation of induced pluripotent stem cells (UCLi024-A) from a patient with argininosuccinate lyase deficiency carrying a homozygous c.437G > A (p.Arg146Gln) mutation.

Argininosuccinic aciduria (ASA) is a rare inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. Patients with ASA present with hyperammonaemia due to an impaired urea cycle pathway in the liver, and systemic disease with epileptic encephalopathy, chronic liver disease, and arterial hypertension. A human induced pluripotent stem cell (iPSC) line from the fibroblasts of a patient with ASA with homozygous pathogenic c.

A live mammalian cells electroporation array for on-chip immunofluorescence.

The detection of intracellular proteins in vitro is commonly realized with immunofluorescence techniques, through which antibodies or markers are delivered into fixed cells and recognize specific proteins. Many innovative techniques, however, avoid cells fixation by chemical compounds and, among the others, electroporation is widely used. Here we demonstrate that in situ electroporation on thin film SiO capacitive microelectrodes can be realized with high efficiency to deliver fluorescent markers and antibodies into mammalian cell lines and primary neuronal cells to detect intracellular proteins, like actin.

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible.

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.

NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform.

The generation of induced pluripotent stem cells (iPSCs) via somatic cell reprogramming allowed to have an unlimited in vitro source of patient-specific cells. This achievement has introduced a new revolutionary way to create human in vitro models and to study human diseases starting from patient's own cells, especially important for inaccessible tissues like the brain. Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology, thanks to the intrinsic high surface-area-to-volume ratio, which allows fine control of the cellular microenvironment.

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.

Atypical CXCL12 signaling enhances neutrophil migration by modulating nuclear deformability.

To reach inflamed tissues from the circulation, neutrophils must overcome physical constraints imposed by the tissue architecture, such as the endothelial barrier or the three-dimensional (3D) interstitial space. In these microenvironments, neutrophils are forced to migrate through spaces smaller than their own diameter. One of the main challenges for cell passage through narrow gaps is the deformation of the nucleus, the largest and stiffest organelle in cells.

A Porous Gelatin Methacrylate-Based Material for 3D Cell-Laden Constructs.

3D constructs are fundamental in tissue engineering and cancer modeling, generating a demand for tailored materials creating a suitable cell culture microenvironment and amenable to be bioprinted. Gelatin methacrylate (GelMA) is a well-known functionalized natural polymer with good printability and binding motifs allowing cell adhesion; however, its tight micropores induce encapsulated cells to retain a non-physiological spherical shape. To overcome this problem, blended GelMa is here blended with Pluronic F-127 (PLU) to modify the hydrogel internal porosity by inducing the formation of larger mesoscale pores.

Timely delivery of cardiac mmRNAs in microfluidics enhances cardiogenic programming of human pluripotent stem cells.

In the last two decades lab-on-chip models, specifically heart-on-chip, have been developed as promising technologies for recapitulating physiological environments suitable for studies of drug and environmental effects on either human physiological or patho-physiological conditions. Most human heart-on-chip systems are based on integration and adaptation of terminally differentiated cells within microfluidic context. This process requires prolonged procedures, multiple steps, and is associated with an intrinsic variability of cardiac differentiation.

Human Pluripotent Stem Cell-Derived Micropatterned Ectoderm Allows Cell Sorting of Meso-Endoderm Lineages.

The human developmental processes during the early post-implantation stage instruct the specification and organization of the lineage progenitors into a body plan. These processes, which include patterning, cell sorting, and establishment of the three germ layers, have been classically studied in non-human model organisms and only recently, through micropatterning technology, in a human-specific context. Micropatterning technology has unveiled mechanisms during patterning and germ layer specification; however, cell sorting and their segregation in specific germ layer combinations have not been investigated yet in a human-specific system.

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.

Derivation and Differentiation of Human Pluripotent Stem Cells in Microfluidic Devices.

An integrative approach based on microfluidic design and stem cell biology enables capture of the spatial-temporal environmental evolution underpinning epigenetic remodeling and the morphogenetic process. We examine the body of literature that encompasses microfluidic applications where human induced pluripotent stem cells are derived starting from human somatic cells and where human pluripotent stem cells are differentiated into different cell types. We focus on recent studies where the intrinsic features of microfluidics have been exploited to control the reprogramming and differentiation trajectory at the microscale, including the capability of manipulating the fluid velocity field, mass transport regime, and controllable composition within micro- to nanoliter volumes in space and time.

The emergence of the circadian clock network in hiPSC-derived hepatocytes on chip.

The circadian clock has paramount implications in physiology and pathology. Although the circadian clock has been widely investigated in adults, up to now very little is known about how circadian rhythms emerge during embryonic development. Some studies about the ontology of the circadian system are focused on the development of the central pacemaker, whereas there is still no agreement about the development of the circadian clock in peripheral tissues.

Using Microfluidics to Generate Human Naïve and Primed Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naïve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations.

SARS-CoV-2 infection and replication in human gastric organoids.

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach.

Synchronization between peripheral circadian clock and feeding-fasting cycles in microfluidic device sustains oscillatory pattern of transcriptome.

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues.

MYOD modified mRNA drives direct on-chip programming of human pluripotent stem cells into skeletal myocytes.

Drug screening and disease modelling for skeletal muscle related pathologies would strongly benefit from the integration of myogenic cells derived from human pluripotent stem cells within miniaturized cell culture devices, such as microfluidic platform. Here, we identified the optimal culture conditions that allow direct differentiation of human pluripotent stem cells in myogenic cells within microfluidic devices. Myogenic cells are efficiently derived from both human embryonic (hESC) or induced pluripotent stem cells (hiPSC) in eleven days by combining small molecules and non-integrating modified mRNA (mmRNA) encoding for the master myogenic transcription factor MYOD.

mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities.

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches.

Development of a microfluidic approach for the real-time analysis of extrinsic TGF-β signalling.

Autocrine and paracrine signalling are traditionally difficult to study due to the sub-micromolar concentrations involved. This has proven to be especially limiting in the study of embryonic stem cells that rely on such signalling for viability, self-renewal, and proliferation. Microfluidics allows to achieve local concentrations of ligands representative of the in vivo stem cell niche, gaining more precise control over the cell microenvironment, as well as to manipulate ligands availability with high temporal resolution and minimal amount of reagents.

In vitro metabolic zonation through oxygen gradient on a chip.

Among the multiple metabolic signals involved in the establishment of the hepatic zonation, oxygen could play a key role. Indeed, depending on hepatocyte position in the hepatic lobule, gene expression and metabolism are differently affected by the oxygen gradient present across the lobule. The aim of this study is to understand whether an oxygen gradient, generated in vitro in our developed device, is sufficient to instruct a functional metabolic zonation during the differentiation of human embryonic stem cells (hESCs) from endoderm toward terminally differentiated hepatocytes, thus mimicking the in vivo situation.

Microfluidic reprogramming to pluripotency of human somatic cells.

Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics.

Direct generation of human naive induced pluripotent stem cells from somatic cells in microfluidics.

Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics.

Cross-talk of healthy and impaired human tissues for dissection of disease pathogenesis.

Systemic diseases affect multiple tissues that interact with each other within a network difficult to explore at the body level. However, understanding the interdependences between tissues could be of high relevance for drug target identification, especially at the first stages of disease development. In vitro systems have the advantages of accessibility to measurements and precise controllability of culture conditions, but currently have limitations in mimicking human in vivo systemic tissue response.

Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures.

Introduction: Epidemiological studies suggest that statins may promote the development or exacerbation of diabetes, but whether this occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which are non-physiologic and have limited clinical transferability.

Methods: Here, we study the effect of simvastatin on insulin secretion using single-islet cultures, an optimal compromise between biological observability and physiologic fidelity.