Human iPSC-derived cerebral organoids model features of Leigh syndrome and reveal abnormal corticogenesis.

Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100 days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.

Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.

Utilizing hiPSC-derived oligodendrocytes to study myelin pathophysiology in neuropsychiatric and neurodegenerative disorders.

Oligodendrocytes play a crucial role in our central nervous system (CNS) by myelinating axons for faster action potential conduction, protecting axons from degeneration, structuring the position of ion channels, and providing nutrients to neurons. Oligodendrocyte dysfunction and/or dysmyelination can contribute to a range of neurodegenerative diseases and neuropsychiatric disorders such as Multiple Sclerosis (MS), Leukodystrophy (LD), Schizophrenia (SCZ), and Autism Spectrum Disorder (ASD). Common characteristics identified across these disorders were either an inability of oligodendrocytes to remyelinate after degeneration or defects in oligodendrocyte development and maturation. Unfortunately, the causal mechanisms of oligodendrocyte dysfunction are still uncertain, and therapeutic targets remain elusive. Many studies rely on the use of animal models to identify the molecular and cellular mechanisms behind these disorders, however, such studies face species-specific challenges and therefore lack translatability. The use of human induced pluripotent stem cells (hiPSCs) to model neurological diseases is becoming a powerful new tool, improving our understanding of pathophysiology and capacity to explore therapeutic targets. Here, we focus on the application of hiPSC-derived oligodendrocyte model systems to model disorders caused by oligodendrocyte dysregulation.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling.

Neural organoids derived from human induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have instead been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional (2D) cultures of human cells, the latter not recapitulating the architecture and cellular diversity of the tissue. To overcome this, we developed a co-culture system of neural organoids generated from human iPSCs fused with fetal leptomeninges from mice with fluorescently labeled meninges (Col1a1-GFP). These proof-of-concept studies test the stability of the different cell types in the leptomeninges (fibroblast and macrophage) and the fused brain organoid (progenitor and neuron), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 days and 60 days in culture, describe best practices for preparing the meninges sample prior to fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.

Revealing the Impact of Mitochondrial Fitness During Early Neural Development Using Human Brain Organoids.

Mitochondrial homeostasis -including function, morphology, and inter-organelle communication- provides guidance to the intrinsic developmental programs of corticogenesis, while also being responsive to environmental and intercellular signals. Two- and three-dimensional platforms have become useful tools to interrogate the capacity of cells to generate neuronal and glia progeny in a background of metabolic dysregulation, but the mechanistic underpinnings underlying the role of mitochondria during human neurogenesis remain unexplored. Here we provide a concise overview of cortical development and the use of pluripotent stem cell models that have contributed to our understanding of mitochondrial and metabolic regulation of early human brain development. We finally discuss the effects of mitochondrial fitness dysregulation seen under stress conditions such as metabolic dysregulation, absence of developmental apoptosis, and hypoxia; and the avenues of research that can be explored with the use of brain organoids.

Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration.

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1-dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1-deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5-mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.

A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.

Uncovering cell biology in the third dimension.

Developmental biology has long benefited from studies of classic model organisms. These model systems have provided the fundamental understanding of general principles of development, as well as insight into genes and signaling pathways that control unique aspects of cell fate specification and tissue morphogenesis. Because human brain development cannot be studied in vivo, scientists have relied on these model systems to study basic principles underlying the development of this complex organ as many of these genes and signaling pathways play conserved roles in human development. However, recent studies have shown species-specific signatures in neurodevelopment such as the transcriptome of outer-radial glia, suggesting use of a human-derived model remains imperative. Over the past decade, human stem cell-derived brain organoids have emerged as a biologically relevant model system to study normal human brain development and neurological diseases. Here, we provide a historical perspective of this emerging model system, discuss current systems and limitations, and propose that new mechanistic insight into cell biology can be revealed using these three-dimensional brain structures.

Modeling the function of BAX and BAK in early human brain development using iPSC-derived systems.

Intrinsic apoptosis relies on the ability of the BCL-2 family to induce the formation of pores on the outer mitochondrial membrane. Previous studies have shown that both BAX and BAK are essential during murine embryogenesis, and reports in human cancer cell lines identified non-canonical roles for BAX and BAK in mitochondrial fission during apoptosis. BAX and BAK function in human brain development remains elusive due to the lack of appropriate model systems. Here, we generated BAX/BAK double knockout human-induced pluripotent stem cells (hiPSCs), hiPSC-derived neural progenitor cells (hNPCs), neural rosettes, and cerebral organoids to uncover the effects of BAX and BAK deletion in an in vitro model of early human brain development. We found that BAX and BAK-deficient cells have abnormal mitochondrial morphology and give rise to aberrant cortical structures. We suggest crucial functions for BAX and BAK during human development, including maintenance of homeostatic mitochondrial morphology, which is crucial for proper development of progenitors and neurons of the cortex. Human pluripotent stem cell-derived systems can be useful platforms to reveal novel functions of the apoptotic machinery in neural development.

Spin∞: an updated miniaturized spinning bioreactor design for the generation of human cerebral organoids from pluripotent stem cells.

Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have become a powerful system to study early development events and to model human disease. Cerebral organoids are generally produced in static culture or in a culture vessel with active mixing, and the two most widely used systems for mixing are a large spinning flask and a miniaturized multi-well spinning bioreactor (also known as Spin Omega (SpinΩ)). The SpinΩ provides a system that is amenable to drug testing, has increased throughput and reproducibility, and utilizes less culture media. However, technical limitations of this system include poor stability of select components and an elevated risk of contamination due to the inability to sterilize the device preassembled. Here, we report a new design of the miniaturized bioreactor system, which we term Spinfinity (Spin∞) that overcomes these concerns to permit long-term experiments. This updated device is amenable to months-long (over 200 days) experiments without concern of unexpected malfunctions.

Wnt Signaling and Its Impact on Mitochondrial and Cell Cycle Dynamics in Pluripotent Stem Cells.

The core transcriptional network regulating stem cell self-renewal and pluripotency remains an intense area of research. Increasing evidence indicates that modified regulation of basic cellular processes such as mitochondrial dynamics, apoptosis, and cell cycle are also essential for pluripotent stem cell identity and fate decisions. Here, we review evidence for Wnt regulation of pluripotency and self-renewal, and its connections to emerging features of pluripotent stem cells, including (1) increased mitochondrial fragmentation, (2) increased sensitivity to cell death, and (3) shortened cell cycle. We provide a general overview of the stem cell-specific mechanisms involved in the maintenance of these uncharacterized hallmarks of pluripotency and highlight potential links to the Wnt signaling pathway. Given the physiological importance of stem cells and their enormous potential for regenerative medicine, understanding fundamental mechanisms mediating the crosstalk between Wnt, organelle-dynamics, apoptosis, and cell cycle will be crucial to gain insight into the regulation of stemness.

A Non-apoptotic Function of MCL-1 in Promoting Pluripotency and Modulating Mitochondrial Dynamics in Stem Cells.

Human pluripotent stem cells (hPSCs) maintain a highly fragmented mitochondrial network, but the mechanisms regulating this phenotype remain unknown. Here, we describe a non-cell death function of the anti-apoptotic protein, MCL-1, in regulating mitochondrial dynamics and promoting pluripotency of stem cells. MCL-1 is induced upon reprogramming, and its inhibition or knockdown induces dramatic changes to the mitochondrial network as well as loss of the key pluripotency transcription factors, NANOG and OCT4. Aside from localizing at the outer mitochondrial membrane like other BCL-2 family members, MCL-1 is unique in that it also resides at the mitochondrial matrix in pluripotent stem cells. Mechanistically, we find MCL-1 to interact with DRP-1 and OPA1, two GTPases responsible for remodeling the mitochondrial network. Depletion of MCL-1 compromised the levels and activity of these key regulators of mitochondrial dynamics. Our findings uncover an unexpected, non-apoptotic function for MCL-1 in the maintenance of mitochondrial structure and stemness.

Apical polarization and lumenogenesis: The apicosome sheds new light.

Establishment of apico-basal polarity is critical for the lumenal epiblast-like morphogenesis of human pluripotent stem cells (hPSCs). In this issue, Taniguchi et al. (2017. J Cell Biol. https://doi.org/10.1083.jcb201704085) describe a structure called the apicosome, generated in single hPSCs, that allows them to self-organize and form the lumenal epiblast-like stage.

Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma.

TGFß signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells.In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis.

Estudio del efecto citoprotector y proliferativo del extracto hidroalcohólico de semillas de mucuna urens sobre un cultivo in vitro de células PC-12 tratadas con dosis crecientes de 6-hidroxidopamina / Study of the cytoprotective and proliferative effect of the hydroalcoholic extract of mucuna urens seeds on an in vitro culture of PC-12 cells treated with increasing doses of 6-hydroxydopamine

La enfermedad de Parkinson es una patología neurodegenerativa producida por la pérdida progresiva de neuronas dopaminérgicas de la sustancia negra y la disfunción de los circuitos neuronales relacionados con el control de los movimientos. Mucuna urens es una planta (Fabaceae) cuya semillas contienen altas concentraciones de levodopa, utilizada desde hace más de 5000 años para el tratamiento de enfermedades como el Parkinson. El objetivo del presente trabajo fue determinar el efecto citoprotector y proliferativo de diversas concentraciones del extracto hidroalcohólico de las semillas de M. urens sobre un cultivo in vitro de células PC-12 tratadas con dosis crecientes de 6-OHDA. Entre los principales hallazgos obtenidos se encuentra una proliferación considerable (superior a 30% sobre el control positivo) de las células incubadas a concentraciones de 0.05 y 1mg/mL del extracto. Además, se observó un efecto de citoprotección, presumiblemente de carácter antioxidante, en células preincubadas con 0.25mg/mL de extracto ante concentraciones de 0.20 y 0.30 mmol/L de neurotoxina. Con este estudio se logró comprobar la capacidad proliferativa y citoprotectora in vitro del extracto hidroalcohólico de M. urens; el cual podría tener implicaciones en tanto en el tratamiento regenerativo como en el preventivo; respectivamente. Así mismo se logró estandarizar el modelo experimental de la línea celular PC-12, ampliamente utilizada a nivel internacional para la investigación de los mecanismos moleculares y celulares de las enfermedades neurodegenerativas y la búsqueda de nuevos tratamientos contra las mismas.