miRNA-based rapid differentiation of purified neurons from hPSCs advancestowards quick screening for neuronal disease phenotypes in vitro

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene

Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy

Role of Human-Induced Pluripotent Stem Cell-Derived Spinal Cord Astrocytes in the Functional Maturation of Motor Neurons in a Multielectrode Array System

The ability to generate human-induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real-time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC-MN maturation in a time-dependent and species-specific manner and suggest a closer approximation of in vivo conditions

Microphysiological Systems for the Evaluation of Biomaterials in Regenerative Therapies

[eng] The design of bioresponsive materials capable of stimulating the body’s innate regenerative potential is opening unprecedented possibilities to treat tissue and organ failure, which is one of the most important burdens of healthcare systems worldwide. Unfortunately, their development is hampered by the lack of adequate preclinical models, which are essential in the successful transition of a biomaterial to the clinical trials phase. Most of the experiments rely on animal models, which usually fail to predict the material interactions with the human body, as they are unable to recapitulate the complexities of our physiology. During the last decades, the advancements in the field of microtechnology have allowed to create advanced cell culture systems capable of replicating tissue and organ-level physiology by mimicking relevant conditions such as cell organization or microenvironmental cues. These platforms, known as microphysiological systems (MPS), have shown in different studies their great potential in predicting mechanisms of action, safety, and efficacy of different drugs, attracting a lot of attention from the pharmaceutical industry and regulatory agencies. However, few studies have explored the possibility of using microphysiological systems for the preclinical testing of biomaterials. The goal of this thesis is to fill this knowledge gap by developing microfluidic cell culture systems that allow to reliably predict the actual in vivo response of different materials. One of the proposed platforms is aimed at assessing the potential of a biomaterial to stimulate endothelial progenitor cell recruitment in a bone tissue microenvironment. This is a critical step in the neovascularization and bone regeneration process that has not been properly studied due to the lack of adequate models. The proposed device allowed to identify the role of calcium ions in stimulating the recruitment of rat endothelial progenitor cells (rEPC) to the site of injury, which is mediated by an increase in the release of osteopontin, a chemotactic and mitogenic protein produced by rat bone-marrow mesenchymal stromal cells (BM-rMSC). The platform was also used to evaluate a calcium-releasing biomaterial based on electrospun polylactic acid (PLA) fibers with calcium-phosphate (CaP) nanoparticles. The results show a significant increase in terms of rEPC recruitment and the release of osteopontin and other pro-angiogenic and inflammatory proteins by BM-rMSC with respect to a regular PLA control, which is in close agreement with previous experiments performed in a murine in vivo model. The other platform proposed in this thesis is aimed at providing a physiologically relevant model of cardiac tissue to study a myocardial ischemia-reperfusion injury. There are currently no reliable in vitro models to mimic this disease, making these contributions extremely relevant for cardiac regeneration studies. A first prototype of the platform based on the combination of aligned electrospun PLA fibers with a user-friendly electrical stimulation setup in a microfluidic cell culture platform produced a biomimetic cardiac tissue in 2D. This was confirmed by the high anisotropy of the tissue constructs, based on the co- culture of neonatal mouse cardiomyocytes with cardiac fibroblasts, as well as the upregulation of several key cardiac markers such as contractile and structural proteins. In order to make the model more physiologically relevant, a second device was developed to obtain human-derived 3D tissues. This platform is based on the self-assembling of primary cardiac fibroblasts (hCF) co-cultured with human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) in a fibrin-based hydrogel around two microposts structures, which exert a passive mechanical tension that stimulates tissue maturation and cell alignment. We first performed a screening using 2D assays based on hPSC-CM monolayers to select the best environmental conditions to mimic an ischemia-reperfusion injury. We then characterized the response of the human- derived cardiac organoids to an ischemia-reperfusion injury, consisting of an 8 h culture period at 0 % oxygen in an ischemic solution that replicates the acidic and hyperkalemic conditions observed in vivo, followed by a refreshment with fully supplemented cell media and recovery of 21 % environmental oxygen concentrations. We observed a drastic increase in cell death by necrosis and apoptosis as well as a strong fibrotic response, characterized by an increase in hCF proliferation, differentiation towards myofibroblasts and collagen I deposition. Taken together, we believe that the platforms developed in this thesis constitute an extremely valuable and versatile tool to perform preclinical studies, offering a promising alternative to animal studies for the development of new biomaterials and drug discovery.[spa] El diseño de biomateriales capaces de estimular la capacidad innata del cuerpo de regenerarse está abriendo una oportunidad sin precedentes para el tratamiento y reemplazamiento de órganos y tejidos, una de las principales cargas en los sistemas de salud a nivel mundial. Desafortunadamente, el desarrollo de estas terapias se ve lastrado por la falta de modelos preclínicos adecuados, que son esenciales en la transición exitosa de un biomaterial a la aplicación clínica. La mayoría de estos experimentos se basan en el uso de modelos animales, que habitualmente fallan en la predicción de las interacciones que ocurren en el cuerpo humano, debido a las diferencias inherentes que existen en términos de fisiología. Durante las últimas décadas, los avances en el campo de la microtecnología han permitido crear plataformas de cultivo celular capaces de replicar elementos fisiológicos a nivel de tejidos y órganos denominados sistemas microfisiológicos. Estos sistemas han demostrado su gran utilidad en la predicción de mecanismos de acción, seguridad y eficacia de diferentes fármacos, atrayendo una gran atención por parte de las agencias regulatorias. Sin embargo, pocos estudios han explorado la posibilidad de usar este tipo de sistemas para la evaluación preclínica de biomateriales. El objetivo de esta tesis es realizar contribuciones en este campo mediante el desarrollo de sistemas microfluídicos de cultivo celular capaces de predecir la respuesta in vivo de diferentes materiales. En esta tesis se presentan principalmente dos modelos diferentes de sistemas microfisiológicos. El primer está relacionado con el reclutamiento de células progenitoras endoteliales en un entorno de regeneración ósea para el estudio de la vascularización de biomateriales, mientras que el segundo busca generar un modelo de tejido cardíaco fisiológicamente relevante para estudiar una lesión por isquemia-reperfusión y posibles terapias regenerativas

Microfluidic devices for cell cultivation and proliferation

Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined

The paroxysmal disorder gene PRRT2 downregulates NaV channels and neuronal excitability in human neurons

Proline-Rich Transmembrane Protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes, including benign familial infantile seizures, paroxysmal kinesigenic dyskinesia and migraine. Most of the mutations of this gene lead to a premature stop codon, generating an unstable form of mRNA or a truncated protein that is degraded, pointing out the loss of the PRRT2 function as pathogenic mechanism of action. In this thesis, we have used different approaches to investigate the pathophysiological function of PRRT2. An important role for PRRT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered by a previous work performed in our laboratory by acute silencing of PRRT2 expression. Here, we analyzed the phenotype of primary hippocampal neurons obtained from mouse PRRT2 knockout (KO) embryos. Analysis of synaptic function in primary neurons obtained from PRRT2-KO showed a largely similar, albeit attenuated, synaptic phenotype with respect to acute PRRT2 silencing characterized by weakened spontaneous/evoked synaptic transmission and increased facilitation at excitatory synapses. These effects were accompanied by a strengthened inhibitory transmission that, however, displayed faster synaptic depression. At the network level, these synaptic phenotypes, resulted in a state of increased spontaneous and evoked neurotransmitter release with increased excitability of excitatory neurons. To better dissect the physiological role of PRRT2, we characterized the phenotypes of neurons differentiated from Induced Pluripotent Stem Cells (iPSCs) from patients homozygous for the PRRT2 c.649dupC mutation. Hence, we observed an increased Na+ current and firing activity in iPSCs rescued with the re-expression of the human wild-type form of PRRT2. By use of heterologous expression system, we demonstrate that PRRT2 interacts with NaV1.2/NaV1.6, but not with NaV1.1 channels, modulating their membrane exposure and decreasing their conductances. In brief, our findings highlighted that PRRT2 mutations might be a negative modulator of NaV1.2/NaV1.6 channels and point out the critical role of this protein in the regulation of the neuronal network functionality

Toward three-dimensional in vitro models to study neurovascular unit functions in health and disease

The high metabolic demands of the brain require an efficient vascular system to be coupled with neural activity to supply adequate nutrients and oxygen. This supply is coordinated by the action of neurons, glial and vascular cells, known collectively as the neurovascular unit, which temporally and spatially regulate local cerebral blood flow through a process known as neurovascular coupling. In many neurodegenerative diseases, changes in functions of the neurovascular unit not only impair neurovascular coupling but also permeability of the blood-brain barrier, cerebral blood flow and clearance of waste from the brain. In order to study disease mechanisms, we need improved physiologically-relevant human models of the neurovascular unit. Advances towards modeling the cellular complexity of the neurovascular unit in vitro have been made using stem-cell derived organoids and more recently, vascularized organoids, enabling intricate studies of non-cell autonomous processes. Engineering and design innovations in microfluidic devices and tissue engineering are progressing our ability to interrogate the cerebrovasculature. These advanced models are being used to gain a better understanding of neurodegenerative disease processes and potential therapeutics. Continued innovation is required to build more physiologically-relevant models of the neurovascular unit encompassing both the cellular complexity and designed features to interrogate neurovascular unit functionality. Keywords: Alzheimer’s disease; cerebrovasculature; in vitro; model; neurodegeneration; neurovascular unit