The Prospects for Retinal Organoids in Treatment of Retinal Diseases.

Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which RO-derived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.

Sensitivity of T1/T2-weighted ratio in detection of cortical demyelination is similar to magnetization transfer ratio using post-mortem MRI.

BACKGROUND: Detecting cortical demyelination using magnetic resonance imaging (MRI) in multiple sclerosis (MS) remains a challenge. Magnetization transfer ratio (MTR), T1-weighted/T2-weighted ratio (T1T2R), and T2-weighted (T2w) signal are sensitive to cortical demyelination, but their accuracy is unknown. OBJECTIVES: To quantify the sensitivity, specificity, and accuracy of postmortem T1T2R, MTR, and T2w in detecting cortical demyelination. METHODS: In situ postmortem MRIs from 9 patients were used to measure T1T2R, MTR, and T2w along the midline of cortical gray matter and classified as normal or abnormal. MRIs were co-registered and compared to hemispheric myelin staining. The sensitivity, specificity, and accuracy of T1T2R, MTR, and T2w in detecting cortical demyelination were measured. RESULTS: The mean age (standard deviation) at death was 64.7 (+/-13.7) years with a disease duration of 23.8 (+/-10.5) years. The sensitivity was 78% for MTR, 75% for T1T2R, and 63% for T2w. The specificity was 46% (T2w), 13% (T1T2R), and 29% (MTR). The accuracy was 71% (T2w), 39% (MTR), and 42% (T1T2R). There were no significant differences between different MRI measures in cortical demyelination or intracortical/subpial lesion detection. CONCLUSIONS: Although somewhat sensitive, the modest specificity of conventional MRI modalities for cortical demyelination indicates that they are influenced by cortical changes other than demyelination. Improved acquisition and post-processing are needed to reliably measure cortical lesion load.

Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy.

Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

T1-/T2-weighted ratio differs in demyelinated cortex in multiple sclerosis.

Detecting cortical demyelination in patients with multiple sclerosis (MS) is difficult. Using magnetic resonance imaging (MRI), ratio maps of T1-weighted (T1w) and T2-weighted (T2w) images may be sensitive to cortical myelin levels. In this MRI-histological study, postmortem in situ scans were acquired from 6 cadavers with MS on a 3T MRI machine. Immunocytochemistry was used to correlate myelin status and cortical T1w/T2w measures. The results showed that the T1w/T2w values significantly differed between demyelinated and myelinated cortex (p < 0.001). The T1w/T2w ratio maps may be a relatively simple, clinically feasible method to assess cortical demyelination. Ann Neurol 2017;82:635-639.

Brain atrophy after bone marrow transplantation for treatment of multiple sclerosis.

BACKGROUND: A cohort of patients with poor-prognosis multiple sclerosis (MS) underwent chemotherapy-based immune ablation followed by immune reconstitution with an autologous hematopoietic stem cell transplant (IA/aHSCT). This eliminated new focal inflammatory activity, but resulted in early acceleration of brain atrophy. OBJECTIVE: We modeled the time course of whole-brain volume in 19 patients to identify the baseline predictors of atrophy and to estimate the average rate of atrophy after IA/aHSCT. METHODS: Percentage whole-brain volume changes were calculated between the baseline and follow-up magnetic resonance imaging (MRI; mean duration: 5 years). A mixed-effects model was applied using two predictors: total busulfan dose and baseline volume of T1-weighted white-matter lesions. RESULTS: Treatment was followed by accelerated whole-brain volume loss averaging 3.3%. Both the busulfan dose and the baseline lesion volume were significant predictors. The atrophy slowed progressively over approximately 2.5 years. There was no evidence that resolution of edema contributed to volume loss. The mean rate of long-term atrophy was -0.23% per year, consistent with the rate expected from normal aging. CONCLUSION: Following IA/aHSCT, MS patients showed accelerated whole-brain atrophy that was likely associated with treatment-related toxicity and degeneration of "committed" tissues. Atrophy eventually slowed to that expected from normal aging, suggesting that stopping inflammatory activity in MS can reduce secondary degeneration and atrophy.

Cuprizone does not induce CNS demyelination in nonhuman primates.

Cognitive decline is a common symptom in multiple sclerosis patients, with profound effects on the quality of life. A nonhuman primate model of multiple sclerosis would be best suited to test the effects of demyelination on complex cognitive functions such as learning and reasoning. Cuprizone has been shown to reliably induce brain demyelination in mice. To establish a nonhuman primate model of multiple sclerosis, young adult cynomolgus monkeys were administered cuprizone per os as a dietary supplement. The subjects received increasing cuprizone doses (0.3-3% of diet) for up to 18 weeks. Magnetic resonance imaging and immunohistological analyses did not reveal demyelination in these monkeys.

Microglial displacement of inhibitory synapses provides neuroprotection in the adult brain.

Microglia actively survey the brain microenvironment and play essential roles in sculpting synaptic connections during brain development. While microglial functions in the adult brain are less clear, activated microglia can closely appose neuronal cell bodies and displace axosomatic presynaptic terminals. Microglia-mediated stripping of presynaptic terminals is considered neuroprotective, but the cellular and molecular mechanisms are poorly defined. Using 3D electron microscopy, we demonstrate that activated microglia displace inhibitory presynaptic terminals from cortical neurons in adult mice. Electrophysiological recordings further establish that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in γ-frequency band. Increased neuronal activity results in the calcium-mediated activation of CaM kinase IV, phosphorylation of CREB, increased expression of antiapoptotic and neurotrophic molecules and reduced apoptosis of cortical neurons following injury. These results indicate that activated microglia can protect the adult brain by migrating to inhibitory synapses and displacing them from cortical neurons.

Association between pathological and MRI findings in multiple sclerosis.

The identification of pathological processes that could be targeted by therapeutic interventions is a major goal of research into multiple sclerosis (MS). Pathological assessment is the gold standard for such identification, but has intrinsic limitations owing to the limited availability of autopsy and biopsy tissue. MRI has gained a leading role in the assessment of MS because it allows doctors to obtain an ante mortem picture of the degree of CNS involvement. A number of correlative pathological and MRI studies have helped to define in vivo the pathological substrates of MS in focal lesions and normal-appearing white matter, not only in the brain, but also in the spinal cord. These studies have resulted in the identification of aspects of pathophysiology that were previously neglected, including grey matter involvement and vascular pathology. Despite these important achievements, numerous open questions still need to be addressed to resolve controversies about how the pathology of MS results in fixed neurological disability.

Advanced MRI in multiple sclerosis: current status and future challenges.

MRI has rapidly become a leading research tool in the study of multiple sclerosis (MS). Conventional imaging is useful in diagnosis and management of the inflammatory stages of MS but has limitations in describing the degree of tissue injury and cause of progressive disability seen in later stages. Advanced MRI techniques hold promise for filling this void. These imaging tools hold great promise to increase understanding of MS pathogenesis and provide greater insight into the efficacy of new MS therapies.

Hematopoietic stem cell transplantation for multiple sclerosis: collaboration of the CIBMTR and EBMT to facilitate international clinical studies.

Clinical investigation of autologous hematopoietic stem cell transplantation (HSCT) as therapy for multiple sclerosis (MS) has been ongoing for over a decade. While several phase II studies have been finalized or are in progress, no definitive prospective randomized studies comparing HSCT versus alternative therapies for MS have been completed. In this conference report of North American and European experts who are involved in the care of MS patients, including neurologists and HSCT physicians, and representatives of the Center for International Blood and Marrow Transplant Research (CIBMTR) and European Group for Blood and Marrow Transplantation (EBMT), we (1) critically review progress to date in HSCT for MS; (2) describe current registry based projects including long-term follow-up studies in HSCT for MS and harmonization of the MS disease-specific research forms that will be used in future by both databases; (3) discuss challenges in study design for a prospective randomized clinical trial of HSCT versus alternative therapy for MS such as feasibility, and the importance of multidisciplinary clinical teams, need for a large sample size and duration of observation required for outcomes assessment; and (4) address future directions in HSCT therapy for MS. To undertake a definitive multicenter clinical trial in autologous HSCT for MS, it will be important to begin well in advance to assemble the team, evaluate proposals for study design, and consider options for the infrastructure and logistical support that will be needed. International collaboration, including partnership with the CIBMTR and EBMT, may be desirable and may in fact be critical for successful completion of a definitive comparative study.

Magnetization transfer ratio evolution with demyelination and remyelination in multiple sclerosis lesions.

OBJECTIVE: To assess demyelination and remyelination in vivo in acute gadolinium (Gd)-enhancing lesions of multiple sclerosis (MS). METHODS: We measured significant changes in magnetization transfer ratio (MTR) consistent with demyelination and remyelination of individual lesion voxels, as well as the mean normalized MTR over all lesion voxels during and after contrast enhancement, in MS patients participating in a 3-year Canadian trial assessing immunoablation and autologous stem cell transplantation for treatment of MS. RESULTS: The average mean normalized lesion MTR over all lesions exhibited partial recovery over 2 to 4 months after Gd enhancement. Voxel-based analysis demonstrated that approximately 70% of the initially enhancing lesion volume (GdLV) was left with stably low MTR over 39 months of evaluation. The percentage of the GdLV undergoing significant increases in MTR consistent with remyelination increased for approximately 7 months after enhancement and then stabilized at 21 %GdLV. Significant decreases in MTR consistent with demyelination were ongoing for approximately 33 months after enhancement, stabilizing at 9 %GdLV. The estimated error of these measurements, based on scan/rescan analysis, was less than 0.4 %GdLV. INTERPRETATION: We found significant changes in MTR consistent with demyelination and remyelination that followed different temporal evolutions and were ongoing in different lesion regions for at least 3 years after lesion formation.