Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.     

Reversal of the Inflammatory Responses in Fabry Patient iPSC-Derived Cardiovascular Endothelial Cells by CRISPR/Cas9-Corrected Mutation

The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.     

Is Inflammation a Friend or Foe for Orthodontic Treatment?: Inflammation in Orthodontically Induced Inflammatory Root Resorption and Accelerating Tooth Movement

The aim of this paper is to provide a review on the role of inflammation in orthodontically induced inflammatory root resorption (OIIRR) and accelerating orthodontic tooth movement (AOTM) in orthodontic treatment. Orthodontic tooth movement (OTM) is stimulated by remodeling of the periodontal ligament (PDL) and alveolar bone. These remodeling activities and tooth displacement are involved in the occurrence of an inflammatory process in the periodontium, in response to orthodontic forces. Inflammatory mediators such as prostaglandins (PGs), interleukins (Ils; IL-1, -6, -17), the tumor necrosis factor (TNF)-α superfamily, and receptor activator of nuclear factor (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) are increased in the PDL during OTM. OIIRR is one of the accidental symptoms, and inflammatory mediators have been detected in resorbed roots, PDL, and alveolar bone exposed to heavy orthodontic force. Therefore, these inflammatory mediators are involved with the occurrence of OIIRR during orthodontic tooth movement. On the contrary, regional accelerating phenomenon (RAP) occurs after fractures and surgery such as osteotomies or bone grafting, and bone healing is accelerated by increasing osteoclasts and osteoblasts. Recently, tooth movement after surgical procedures such as corticotomy, corticision, piezocision, and micro-osteoperforation might be accelerated by RAP, which increases the bone metabolism. Therefore, inflammation may be involved in accelerated OTM (AOTM). The knowledge of inflammation during orthodontic treatment could be used in preventing OIIRR and AOTM.     

A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.     

The Presence of Blood–Brain Barrier Modulates the Response to Magnesium Salts in Human Brain Organoids

Magnesium (Mg) is fundamental in the brain, where it regulates metabolism and neurotransmission and protects against neuroinflammation. To obtain insights into the molecular basis of Mg action in the brain, we investigated the effects of Mg in human brain organoids, a revolutionary 3D model to study neurobiology and neuropathology. In particular, brain organoids derived from human induced pluripotent stem cells were cultured in the presence or in the absence of an in vitro-generated blood–brain barrier (BBB), and then exposed to 1 or 5 mM concentrations of inorganic and organic Mg salts (Mg sulphate (MgSO₄); Mg pidolate (MgPid)). We evaluated the modulation of NMDA and GABAergic receptors, and BDNF. Our data suggest that the presence of the BBB is essential for Mg to exert its effects on brain organoids, and that 5 mM of MgPid is more effective than MgSO₄ in increasing the levels of GABA receptors and BDNF, and decreasing those of NMDA receptor. These results might illuminate novel pathways explaining the neuroprotective role of Mg.     

Pannexin 1 Transgenic Mice: Human Diseases and Sleep-Wake Function Revision

In humans and other vertebrates pannexin protein family was discovered by homology to invertebrate gap junction proteins. Several biological functions were attributed to three vertebrate pannexins members. Six clinically significant independent variants of the PANX1 gene lead to human infertility and oocyte development defects, and the Arg217His variant was associated with pronounced symptoms of primary ovarian failure, severe intellectual disability, sensorineural hearing loss, and kyphosis. At the same time, only mild phenotypes were observed in Panx1 knockout mice. In addition, a passenger mutation was identified in a popular line of Panx1 knockout mice, questioning even those effects. Using CRISPR/Cas9, we created a new line of Panx1 knockout mice and a new line of mice with the clinically significant Panx1 substitution (Arg217His). In both cases, we observed no significant changes in mouse size, weight, or fertility. In addition, we attempted to reproduce a previous study on sleep/wake and locomotor activity functions in Panx1 knockout mice and found that previously reported effects were probably not caused by the Panx1 knockout itself. We consider that the pathological role of Arg217His substitution in Panx1, and some Panx1 functions in general calls for a re-evaluation.     

Human iPSC-Derived 2D and 3D Platforms for Rapidly Assessing Developmental,Functional, and Terminal Toxicities in Neural Cells

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.     

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells to Model Myogenesis and Muscle Regeneration

In vitro organoids derived from human pluripotent stem cells (hPSCs) have been developed as essential tools to study the underlying mechanisms of human development and diseases owing to their structural and physiological similarity to corresponding organs. Despite recent advances, there are a few methodologies for three-dimensional (3D) skeletal muscle differentiation, which focus on the terminal differentiation into myofibers and investigate the potential of modeling neuromuscular disorders and muscular dystrophies. However, these methodologies cannot recapitulate the developmental processes and lack regenerative capacity. In this study, we developed a new method to differentiate hPSCs into a 3D human skeletal muscle organoid (hSkMO). This organoid model could recapitulate the myogenesis process and possesses regenerative capacities of sustainable satellite cells (SCs), which are adult muscle stem/progenitor cells capable of self-renewal and myogenic differentiation. Our 3D model demonstrated myogenesis through the sequential occurrence of multiple myogenic cell types from SCs to myocytes. Notably, we detected quiescent, non-dividing SCs throughout the hSkMO differentiation in long-term culture. They were activated and differentiated to reconstitute muscle tissue upon damage. Thus, hSkMOs can recapitulate human skeletal muscle development and regeneration and may provide a new model for studying human skeletal muscles and related diseases.     

Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.     

Available In Vitro Models for Human Satellite Cells from Skeletal Muscle

Skeletal muscle accounts for almost 40% of the total adult human body mass. This tissue is essential for structural and mechanical functions such as posture, locomotion, and breathing, and it is endowed with an extraordinary ability to adapt to physiological changes associated with growth and physical exercise, as well as tissue damage. Moreover, skeletal muscle is the most age-sensitive tissue in mammals. Due to aging, but also to several diseases, muscle wasting occurs with a loss of muscle mass and functionality, resulting from disuse atrophy and defective muscle regeneration, associated with dysfunction of satellite cells, which are the cells responsible for maintaining and repairing adult muscle. The most established cell lines commonly used to study muscle homeostasis come from rodents, but there is a need to study skeletal muscle using human models, which, due to ethical implications, consist primarily of in vitro culture, which is the only alternative way to vertebrate model organisms. This review will survey in vitro 2D/3D models of human satellite cells to assess skeletal muscle biology for pre-clinical investigations and future directions.     

Retinoic Acid Promotes the In Vitro Growth,Patterning and Improves the Cellular Composition of Human Pluripotent Stem-Cell-Derived Intestinal Organoids

Human intestinal organoids (HIOs) generated from human pluripotent stem cells hold great promise for modeling human development and as a possible source of tissue for transplantation. HIOs generate all of the main epithelial and mesenchymal cell types found in the developing human intestine and mature into intestinal tissue with crypts and villi following transplantation into immunocompromised mice. However, incomplete in vitro patterning and the presence of contaminating neurons could hinder their use for regenerative medicine in humans. Based on studies in model organisms, we hypothesized that the treatment of HIOs with all trans retinoic acid (ATRA) would improve their in vitro growth and patterning. We found that ATRA not only improved the patterning of HIOs, ATRA also increased organoid forming efficiency, improved epithelial growth, enriched intestinal subepithelial myofibroblasts (ISEMFs) and reduced neuronal contamination in HIOs. Taken together, our studies demonstrate how the manipulation of a single developmental signaling pathway can be used to improve the survival, patterning and cellular composition of HIOs.     

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.     

Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications

Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.     

The Potential of the Combination of CRISPR/Cas9 and Pluripotent Stem Cells to Provide Human Organs from Chimaeric Pigs

Clinical organ allotransplantation is limited by the availability of deceased human donors. However, the transplantation of human organs produced in other species would provide an unlimited number of organs. The pig has been identified as the most suitable source of organs for humans as organs of any size would be available. Genome editing by RNA-guided endonucleases, also known as clustered regularly interspaced short palindromic repeat (CRISPR/Cas9), in combination with induced pluripotent stem cells (iPSC), may have the potential to enable the creation of human organs from genetically-modified chimaeric pigs. These could potentially provide an unlimited supply of organs that would not be rejected by the recipient’s immune system. However, substantial research is needed to prove that this approach will work. Genetic modification of chimaeric pigs could also provide useful models for developing therapies for various human diseases, especially in relation to drug development.     

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.     

From 2D to 3D Co-Culture Systems: A Review of Co-Culture Models to Study the Neural Cells Interaction

The central nervous system (CNS) controls and regulates the functional activities of the organ systems and maintains the unity between the body and the external environment. The advent of co-culture systems has made it possible to elucidate the interactions between neural cells in vitro and to reproduce complex neural circuits. Here, we classified the co-culture system as a two-dimensional (2D) co-culture system, a cell-based three-dimensional (3D) co-culture system, a tissue slice-based 3D co-culture system, an organoid-based 3D co-culture system, and a microfluidic platform-based 3D co-culture system. We provide an overview of these different co-culture models and their applications in the study of neural cell interaction. The application of co-culture systems in virus-infected CNS disease models is also discussed here. Finally, the direction of the co-culture system in future research is prospected.     

Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds

CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.