Establishment Of Disease Models Research Articles

Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.

Scenario analysis of COVID-19 dynamical variations by different social environmental factors: a case study in Xinjiang.

With the rapid advancement of the One Health approach, the transmission of human infectious diseases is generally related to environmental and animal health. Coronavirus disease (COVID-19) has been largely impacted by environmental factors regionally and globally and has significantly disrupted human society, especially in low-income regions that border many countries. However, few research studies have explored the impact of environmental factors on disease transmission in these regions. We used the Xinjiang Uygur Autonomous Region as the study area to investigate the impact of environmental factors on COVID-19 variation using a dynamic disease model. Given the special control and prevention strategies against COVID-19 in Xinjiang, the focus was on social and environmental factors, including population mobility, quarantine rates, and return rates. The model performance was evaluated using the statistical metrics of correlation coefficient (CC), normalized absolute error (NAE), root mean square error (RMSE), and distance between the simulation and observation (DISO) indices. Scenario analyses of COVID-19 in Xinjiang encompassed three aspects: different population mobilities, quarantine rates, and return rates. The results suggest that the established dynamic disease model can accurately simulate and predict COVID-19 variations with high accuracy. This model had a CC value of 0.96 and a DISO value of less than 0.35. According to the scenario analysis results, population mobilities have a large impact on COVID-19 variations, with quarantine rates having a stronger impact than return rates. These results provide scientific insight into the control and prevention of COVID-19 in Xinjiang, considering the influence of social and environmental factors on COVID-19 variation. The control and prevention strategies for COVID-19 examined in this study may also be useful for the control of other infectious diseases, especially in low-income regions that are bordered by many countries.

Gabapentin improves neuropathic pain in Minamata disease model rats.

Methylmercury (MeHg), the causative agent of Minamata disease, damages the cranial nervous system and causes specific sensory disturbances, especially hypoesthesia, in the extremities. However, recent reports demonstrate that patients with chronic Minamata disease conversely develop neuropathic pain in the lower extremities. Studies on our established Minamata disease model rats showed that MeHg-mediated neurodegeneration might induce neuropathic pain by over time through inducing rewiring with neuronal activation in the somatosensory cortex via microglial activation in the spinal dorsal horn. In this study, the effects of gabapentin, a potentially effective treatment for neuropathic pain, was evaluated using this Minamata disease model rats. To further elucidate the mechanism of its medicinal effects, histochemical and biochemical analyses of the nervous system of Minamata disease model rats were conducted. Gabapentin treatment restored the reduction in the pain threshold caused by MeHg exposure in rats. Histochemical and biochemical analyses revealed that gabapentin showed no effect on MeHg-induced neurodegeneration in entire nervous system and microglial activation in the spinal dorsal horn. However, it was shown that gabapentin may reduce excessive synaptogenesis through its antagonist action on the alpha2-delta-1 subunit of calcium channels in the somatosensory cortex. These results indicate that gabapentin may alleviated neuropathic pain in MeHg poisoning, as typified by Minamata disease, by reversibly modulation synaptic rewiring in the somatosensory cortex.

Construction of tumour disease models based on CRISPR/Cas and iPSC technology

Cancer, which is defined as malignant tumours and neoplasms, is one of the most concerned diseases in the world. According to the WHO website, almost 10 million of people lost their lives due to cancer in 2020. The complexity of tumorigenesis and cancer development, which have not been explored enough, lead to its high morbidity and mortality. Therefore, it is in an urgent need to establish stable and reliable tumour disease models to study the complicated pathogenesis, hence developing novel therapies and drugs. As the third generation of gene-editing technology, CRISPR/Cas system has the advantages of high suitability to all types of model forms owing to the simplicity and convenience of operation. It can efficiently construct tumour disease models for verifying and screening related targets. iPSC, as a stem cell technology, can provide a new cell source for CRISPR/Cas system-based tumour disease modelling. It can be the basic of establishing disease models in specific areas like brain tumours. This review introduced the advantages of CRISPR/Cas gene editing and iPSC technology in establishing disease models and the combined application strategies of the two technologies through the cases of tumour disease modelling in the past five years. It also explored the modelling strategies and model categories adopted in the popular cancer field.

HDL mimetics protect Alzheimer’s patients carrying APOE ε4 from SARS-CoV-2 invasion

Patients with Alzheimer’s disease (AD) inheriting Apolipoprotein (APOE) ε4 are susceptible to COVID-19, but the underlying mechanism of how such a neurodegenerative disease promotes respiratory vulnerability to viral infections remains insufficiently understood. The uncovering of genetic basis of COVID-19 outcome holds the potential to establish disease models for therapeutic development. Here, using human iPSC-derived type II alveolar epithelial cells (hAECII) from AD patients with APOE ε4 and healthy individuals, we showed that AECII of AD had a greater level of SARS-CoV-2 invasion but not replication, along with increased expressions of viral receptor and co-receptors, while reduced pulmonary surfactant proteins. Since low serum HDL-C levels have been implicated in the onset of both AD and COVID-19, we further revealed that HDL mimetics, including 4 F dimeric peptide and its phospholipid conjugate pHDL, were effective in protecting AECII bearing APOE ε4 against SARS-CoV-2 invasion. In AD, concomitant with improved AD pathological phenotypes, HDL mimetics specifically restored the defective basal SP-D, a pulmonary innate immune lipoprotein that targets glycans of the spike protein to neutralize invading viruses. Moreover, HDL mimetics demonstrated strong SP-D-independent virucidal effects, which additionally restricted the entry of coronaviral particles into non-AD AECII models and lungs of human ACE2-transgenic mice in response to Alpha and Omicron variants of SARS-CoV-2. Our work offers critical insights into the respiratory sensitivity of AD patients carrying APOE ε4 to viral infection and repurpose HDL-based therapeutics as potential preventive interventions in respiratory viral pandemics.

A Mathematical Study on a Fractional-Order SEIR Mpox Model: Analysis and Vaccination Influence

This paper establishes a novel fractional-order version of a recently expanded form of the Susceptible-Exposed-Infectious-Recovery (SEIR) Mpox model. This model is investigated by means of demonstrating some significant findings connected with the stability analysis and the vaccination impact, as well. In particular, we analyze the fractional-order Mpox model in terms of its invariant region, boundedness of solution, equilibria, basic reproductive number, and its elasticity. In accordance with an effective vaccine, we study the progression and dynamics of the Mpox disease in compliance with various scenarios of the vaccination ratio through the proposed fractional-order Mpox model. Accordingly, several numerical findings of the proposed model are depicted with the use of two numerical methods; the Fractional Euler Method (FEM) and Modified Fractional Euler Method (MFEM). Such findings demonstrate the influence of the fractional-order values coupled with the vaccination rate on the dynamics of the established disease model.

Murine Models and Research Progress on Dysmenorrhea.

Dysmenorrhea is a prevalent gynecological disease among women at reproductive age. It is classified as the primary dysmenorrhea and the secondary dysmenorrhea according to the etiology. The primary dysmenorrhea is caused by uterine hypercontraction without any identifiable pelvic lesions, while the secondary dysmenorrhea is incurred by gynecological disorder with pelvic organic lesions. However, the underlying mechanism of dysmenorrhea is not completely clear. Animal models of dysmenorrhea, especially mouse and rat model, are helpful to explore the pathophysiological mechanism of dysmenorrhea, clarify the therapeutic effect of compounds, and guide clinical treatment. The murine model of primary dysmenorrhea is commonly induced by oxytocin or prostaglandin F2α, while the secondary dysmenorrhea murine model was further created by injecting oxytocin on the basis of the established primary disease model. This review summarizes the current progress of dysmenorrhea models in rodent, including experimental methods, corresponding evaluation indexes, and the advantages and disadvantages of various murine dysmenorrhea models, in order to provide a reference for the selection of murine dysmenorrhea models and the further study of the pathophysiological mechanism of dysmenorrhea.

Recipe for pituitary organoids.

Generation of a variety of organs and tissues from human pluripotent stem cells (hPSCs) has been attempted in vitro. We here present a simple and efficient method for induction of hypothalamic and pituitary tissues from hPSCs. On provision of exogenous agents important for early hypothalamus-pituitary organogenesis, including bone morphogenetic protein 4 and activators of sonic hedgehog, in three-dimensional culture, hPSCs spontaneously form spherical organoids with two distinct tissues, hypothalamus and adenohypophysis. The pituitary tissues derived from hPSCs not only secrete adenocorticotropic hormone, but also retain both positive and negative feedback mechanisms, recapitulating mature endocrine organs in vivo. Furthermore, the results of ectopic transplantation with mouse models of hypopituitarism suggest that these hypothalamus-pituitary organoids have potential as engraftment organs. In addition to their use in transplantation for patients with hypopituitarism they will allow establishment of disease models in vitro and enable research impossible in humans. Hypothalamus-pituitary organoids promise to be a powerful tool in regenerative medicine, drug discovery, and basic research into pituitary development.

MODL-01. TRACTABLE PATIENT-DERIVED MODELS FOR PRECLINICAL THERAPEUTIC STUDIES OF CNS METASTASES

Abstract INTRODUCTION Brain metastases are the most common brain tumors and occur in 10-30% of cancer patients, whereas leptomeningeal disease (LMD) occurs in approximately 5% of adults with systemic malignancies. Tractable preclinical disease models that faithfully represent metastasis to the brain and recapitulate LMD are needed to improve our understanding of the biological basis of CNS disease as well developing effective therapeutic strategies. Our goal in this study was to generate representative preclinical disease models using two methods. METHODS We isolated tumor cells from CSF of 16 patients with cytologically proven LMD (9 NSCLC, 1 melanoma, 1 ovarian cancer, 1 endometrial cancer, and 4 breast cancer) and implanted the cells subcutaneously into the flank of immunocompromised mice. Cell lines were also generated from PDX tissues. Models were characterized by next-generation sequencing (NGS). We also generated a model of CNS metastasis of kinase-driven sarcoma by intracardiac (IC) injection of human mesenchymal stem cells (HMSC) expressing a SPECC1L::RET fusion (CRISPR-Cas9 gene editing). The cells (HMSC-RET) were also labeled with a luciferase construct to allow non-invasive bioluminescence imaging. RESULTS We established three PDX models (2 lung, 1 ovarian) from CSF (19% success rate compared to approximately 33% for solid tumors) and matched cell lines from the resulting PDX tissues. Intracardiac injection of HMSC-RET cells resulted in tumors establishing in several peripheral organs and the brain. SUMMARY AND CONCLUSIONS We have established disease models of CNS metastasis and LMD. Translational studies where patients with clinical suspicion of LMD undergo CSF sampling, NGS/ctDNA analysis, and PDX modeling are crucial in improving our understanding of this metastatic compartment and investigating novel treatment paradigms. Future studies will be focused on examining the biochemical and genetic nature of these tumors as well as developing effective therapeutic strategies.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

Current landscape of gene‐editing technology in biomedicine: Applications, advantages, challenges, and perspectives

The expanding genome editing toolbox has revolutionized life science research ranging from the bench to the bedside. These “molecular scissors” have offered us unprecedented abilities to manipulate nucleic acid sequences precisely in living cells from diverse species. Continued advances in genome editing exponentially broaden our knowledge of human genetics, epigenetics, molecular biology, and pathology. Currently, gene editing‐mediated therapies have led to impressive responses in patients with hematological diseases, including sickle cell disease and thalassemia. With the discovery of more efficient, precise and sophisticated gene‐editing tools, more therapeutic gene‐editing approaches will enter the clinic to treat various diseases, such as acquired immunodeficiency sydrome (AIDS), hematologic malignancies, and even severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. These initial successes have spurred the further innovation and development of gene‐editing technology. In this review, we will introduce the architecture and mechanism of the current gene‐editing tools, including clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated nuclease‐based tools and other protein‐based DNA targeting systems, and we summarize the meaningful applications of diverse technologies in preclinical studies, focusing on the establishment of disease models and diagnostic techniques. Finally, we provide a comprehensive overview of clinical information using gene‐editing therapeutics for treating various human diseases and emphasize the opportunities and challenges.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts invitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit invivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n= 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p<0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p<0.001), fibronectin (3.2-fold; p<0.001), collagen I (0.8-fold; p<0.001), and collagen III (1.4-fold; p<0.001), as well as protein levels of α-SMA (23.8%; p<0.001) and collagens (1.8-fold; p<0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-β signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Global Trends of Stem Cell Precision Medicine Research (2018-2022): A Bibliometric Analysis.

BackgroundStem cells are a group of cells that can self-renew and have multiple differentiation capabilities. Shinya Yamanaka first discovered a method to convert somatic cells into pluripotent stem cells in 2006. Stem cell therapy can be summarized into three aspects (regenerative treatment, therapy targeted at stem cells, and establishment of disease models). Disease models are mainly established by induced pluripotent stem cells, and the research of stem cell precision medicine has been promising in recent years. Based on the construction of 3D, patient-specific disease models from pluripotent induced stem cells, proper research on disease development and treatment prognosis can be realized. Bibliometric analysis is an efficient way to quickly understand global trends and hotspots in this field.MethodsA literature search of stem cell precision medicine research from 2018 to 2022 was carried out using the Web of Science Core Collection.VOSviewer, R-bibliometrix, and CiteSpace software programs were employed to perform the bibliometric analysis.ResultsA total of 552 publications were retrieved from 2018 to 2022. Annual publication outputs trended upward and reached a peak of 172 in 2021. The United States contributed the most publications (160, 29.0%) to the field, followed by China (63, 11.4%) and Italy (60, 10.9%). International academic collaborations were active. CANCERS was considered the most productive journal with 18 documents. NATURE was the most co-cited journal with 1860 times citations. The most cited document was entitled “Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association” with 9 times local citations. “ precision medicine” (n = 89, 12.64%), “personalized medicine” (n = 72, 10.23%), “stem cells” (n = 43, 4.40%), and “induced pluripotent stem cells” (n = 41, 5.82%), “cancer stem cells” (n = 31, 4%), “organoids” (n = 26, 3.69%) were the top 6 frequent keywords.ConclusionThe present study performs a comprehensive investigation concerning stem cell precision medicine (2018–2022) for the first time. This research field is developing, and a deeper exploration of 3D patient-specific organoid disease models is worth more research in the future.

Cellular and animal models to investigate pathogenesis of amyloid aggregation in neurodegenerative diseases.

Abnormal aggregation of amyloid proteins, e.g. amyloid β (Aβ), Tau and α-synuclein (α-syn), is closely associated with a variety of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Cellular and animal models are useful to explore the neuropathology of amyloid aggregates in disease initiation and progression. In this protocol, we describe detailed procedures for how to establish neuronal and PD mouse models to evaluate amyloid pathologies including self-propagation, cell-to-cell transmission, neurotoxicity, and impact on mouse motor and cognitive functions. We use α-syn, a key pathogenic protein in PD, as an example to demonstrate the application of the protocol, while it can be used to investigate the pathologies of other amyloid proteins as well. The established disease models are also useful to assess the activities of drug candidates for therapeutics of neurodegenerative diseases.

Mitochondrial Phenotypes in Parkinson’s Diseases—A Focus on Human iPSC-Derived Dopaminergic Neurons

Established disease models have helped unravel the mechanistic underpinnings of pathological phenotypes in Parkinson’s disease (PD), the second most common neurodegenerative disorder. However, these discoveries have been limited to relatively simple cellular systems and animal models, which typically manifest with incomplete or imperfect recapitulation of disease phenotypes. The advent of induced pluripotent stem cells (iPSCs) has provided a powerful scientific tool for investigating the underlying molecular mechanisms of both familial and sporadic PD within disease-relevant cell types and patient-specific genetic backgrounds. Overwhelming evidence supports mitochondrial dysfunction as a central feature in PD pathophysiology, and iPSC-based neuronal models have expanded our understanding of mitochondrial dynamics in the development and progression of this devastating disorder. The present review provides a comprehensive assessment of mitochondrial phenotypes reported in iPSC-derived neurons generated from PD patients’ somatic cells, with an emphasis on the role of mitochondrial respiration, morphology, and trafficking, as well as mitophagy and calcium handling in health and disease. Furthermore, we summarize the distinguishing characteristics of vulnerable midbrain dopaminergic neurons in PD and report the unique advantages and challenges of iPSC disease modeling at present, and for future mechanistic and therapeutic applications.

Fractional-order COVID-19 pandemic outbreak: Modeling and stability analysis

Today, the entire world is witnessing an enormous upsurge in coronavirus pandemic (COVID-19 pandemic). Confronting such acute infectious disease, which has taken multiple victims around the world, requires all specialists in all fields to devote their efforts to seek effective treatment or even control its disseminate. In the light of this aspect, this work proposes two new fractional-order versions for one of the recently extended forms of the SEIR model. These two versions, which are established in view of two fractional-order differential operators, namely, the Caputo and the Caputo–Fabrizio operators, are numerically solved based on the Generalized Euler Method (GEM) that considers Caputo sense, and the Adams–Bashforth Method (ABM) that considers Caputo–Fabrizio sense. Several numerical results reveal the impact of the fractional-order values on the two established disease models, and the continuation of the COVID-19 pandemic outbreak to this moment. In the meantime, some novel results related to the stability analysis and the basic reproductive number are addressed for the proposed fractional-order Caputo COVID-19 model. For declining the total of individuals infected by such pandemic, a new compartment is added to the proposed model, namely the disease prevention compartment that includes the use of face masks, gloves and sterilizers. In view of such modification, it is turned out that the performed addition to the fractional-order Caputo COVID-19 model yields a significant improvement in reducing the risk of COVID-19 spreading.

Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy

SummaryFukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

MPS VI associated ocular phenotypes in an MPS VI murine model and the therapeutic effects of odiparcil treatment

Maroteaux – Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is a lysosomal storage disease resulting from insufficient enzymatic activity for degradation of the specific glycosaminoglycans (GAG) chondroitin sulphate (CS) and dermatan sulphate (DS). Among the most pronounced MPS VI clinical manifestations caused by cellular accumulation of excess CS and DS are eye disorders, in particular those that affect the cornea. Ocular manifestations are not treated by the current standard of care, enzyme replacement therapy (ERT), leaving patients with a significant unmet need. Using in vitro and in vivo models, we previously demonstrated the potential of the β-D-xyloside, odiparcil, as an oral GAG clearance therapy for MPS VI. Here, we characterized the eye phenotypes in MPS VI arylsulfatase B deficient mice (Arsb−) and studied the effects of odiparcil treatment in early and established disease models. Severe levels of opacification and GAG accumulation were detected in the eyes of MPS VI Arsb− mice. Histological examination of MPS VI Arsb− eyes showed an aggregate of corneal phenotypes, including reduction in the corneal epithelium thickness and number of epithelial cell layers, and morphological malformations in the stroma. In addition, colloidal iron staining showed specifically GAG accumulation in the cornea. Orally administered odiparcil markedly reduced GAG accumulation in the eyes of MPS VI Arsb− mice in both disease models and restored the corneal morphology (epithelial layers and stromal structure). In the early disease model of MPS VI, odiparcil partially reduced corneal opacity area, but did not affect opacity area in the established model. Analysis of GAG types accumulating in the MPS VI Arsb− eyes demonstrated major contribution of DS and CS, with some increase in heparan sulphate (HS) as well and all were reduced with odiparcil treatment. Taken together, we further reveal the potential of odiparcil to be an effective therapy for eye phenotypes associated with MPS VI disease.

Physiological Characterization and Transcriptomic Properties of GnRH Neurons Derived From Human Stem Cells.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell–derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.