Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R.
METHODS: Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL.
RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls.
CONCLUSIONS: We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

Neuroinflammation is a complex biological process that plays a significant role in various brain disorders. Microglia and astrocytes are the key cell types involved in inflammatory responses in the central nervous system. Neuroinflammation results in increased levels of secreted inflammatory factors, such as cytokines, chemokines, and reactive oxygen species. To model neuroinflammation in vitro, various human induced pluripotent stem cell (iPSC)-based models have been utilized, including monocultures, transfer of conditioned media between cell types, co-culturing multiple cell types, neural organoids, and xenotransplantation of cells into the mouse brain. To induce neuroinflammatory responses in vitro, several stimuli have been established that can induce responses in either microglia, astrocytes, or both. Here, we describe and critically evaluate the different types of iPSC models that can be used to study neuroinflammation and highlight how neuroinflammation has been induced and measured in these cultures.

UNASSIGNED: Our limited understanding of the mechanisms that trigger the emergence of Alzheimer\'s disease (AD) has contributed to the lack of interventions that stop, prevent, or fully treat this disease. We believe that the development of a non-human primate model of AD will be an essential step toward overcoming limitations of other model systems and is crucial for investigating primate-specific mechanisms underlying the cellular and molecular root causes of the pathogenesis and progression of AD.
UNASSIGNED: A new consortium has been established with funding support from the National Institute on Aging aimed at the generation, characterization, and validation of Marmosets As Research Models of AD (MARMO-AD). This consortium will study gene-edited marmoset models carrying genetic risk for AD and wild-type genetically diverse aging marmosets from birth throughout their lifespan, using non-invasive longitudinal assessments. These include characterizing the genetic, molecular, functional, behavioral, cognitive, and pathological features of aging and AD.
UNASSIGNED: The consortium successfully generated viable founders carrying PSEN1 mutations in C410Y and A426P using CRISPR/Cas9 approaches, with germline transmission demonstrated in the C410Y line. Longitudinal characterization of these models, their germline offspring, and normal aging outbred marmosets is ongoing. All data and resources from this consortium will be shared with the greater AD research community.
UNASSIGNED: By establishing marmoset models of AD, we will be able to investigate primate-specific cellular and molecular root causes that underlie the pathogenesis and progression of AD, overcome limitations of other model organisms, and support future translational studies to accelerate the pace of bringing therapies to patients.

The unprecedented rise in life expectancy observed in the last decades is leading to a global increase in the ageing population, and age-associated diseases became an increasing societal, economic, and medical burden. This has boosted major efforts in the scientific and medical research communities to develop and improve therapies to delay ageing and age-associated functional decline and diseases, and to expand health span. The establishment of induced pluripotent stem cells (iPSCs) by reprogramming human somatic cells has revolutionised the modelling and understanding of human diseases. iPSCs have a major advantage relative to other human pluripotent stem cells as their obtention does not require the destruction of embryos like embryonic stem cells do, and do not have a limited proliferation or differentiation potential as adult stem cells. Besides, iPSCs can be generated from somatic cells from healthy individuals or patients, which makes iPSC technology a promising approach to model and decipher the mechanisms underlying the ageing process and age-associated diseases, study drug effects, and develop new therapeutic approaches. This review discusses the advances made in the last decade using iPSC technology to study the most common age-associated diseases, including age-related macular degeneration (AMD), neurodegenerative and cardiovascular diseases, brain stroke, cancer, diabetes, and osteoarthritis.

Understanding the complexities of the human brain and its associated disorders poses a significant challenge in neuroscience. Traditional research methods have limitations in replicating its intricacies, necessitating the development of in vitro models that can simulate its structure and function. Three-dimensional in vitro models, including organoids, cerebral organoids, bioprinted brain models, and functionalized brain organoids, offer promising platforms for studying human brain development, physiology, and disease. These models accurately replicate key aspects of human brain anatomy, gene expression, and cellular behavior, enabling drug discovery and toxicology studies while providing insights into human-specific phenomena not easily studied in animal models. The use of human-induced pluripotent stem cells has revolutionized the generation of 3D brain structures, with various techniques developed to generate specific brain regions. These advancements facilitate the study of brain structure development and function, overcoming previous limitations due to the scarcity of human brain samples. This technical review provides an overview of current 3D in vitro models of the human cortex, their development, characterization, and limitations, and explores the state of the art and future directions in the field, with a specific focus on their applications in studying neurodevelopmental and neurodegenerative disorders.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Microphysiological systems (MPS) are 2D or 3D multicellular constructs able to mimic tissue microenvironments. The latest models encompass a range of techniques, including co-culturing of various cell types, utilization of scaffolds and extracellular matrix materials, perfusion systems, 3D culture methods, 3D bioprinting, organ-on-a-chip technology, and examination of tissue structures. Several human brain 3D cultures or brain MPS (BMPS) have emerged in the last decade. These organoids or spheroids are 3D culture systems derived from induced pluripotent cells or embryonic stem cells that contain neuronal and glial populations and recapitulate structural and physiological aspects of the human brain. BMPS have been introduced recently in the study and modeling of neuroinfectious diseases and have proven to be useful in establishing neurotropism of viral infections, cell-pathogen interactions needed for infection, assessing cytopathological effects, genomic and proteomic profiles, and screening therapeutic compounds. Here we review the different methodologies of organoids used in neuroinfectious diseases including spheroids, guided and unguided protocols as well as microglia and blood-brain barrier containing models, their specific applications, and limitations. The review provides an overview of the models existing for specific infections including Zika, Dengue, JC virus, Japanese encephalitis, measles, herpes, SARS-CoV2, and influenza viruses among others, and provide useful concepts in the modeling of disease and antiviral agent screening.