Neuroinflammatory disease signatures in SPG11-related hereditary spastic paraplegia patients.

Biallelic loss of SPG11 function constitutes the most frequent cause of complicated autosomal recessive hereditary spastic paraplegia (HSP) with thin corpus callosum, resulting in progressive multisystem neurodegeneration. While the impact of neuroinflammation is an emerging and potentially treatable aspect in neurodegenerative diseases and leukodystrophies, the role of immune cells in SPG11-HSP patients is unknown. Here, we performed a comprehensive immunological characterization of SPG11-HSP, including examination of three human postmortem brain donations, immunophenotyping of patients' peripheral blood cells and patient-specific induced pluripotent stem cell-derived microglia-like cells (iMGL). We delineate a previously unknown role of innate immunity in SPG11-HSP. Neuropathological analysis of SPG11-HSP patient brain tissue revealed profound microgliosis in areas of neurodegeneration, downregulation of homeostatic microglial markers and cell-intrinsic accumulation of lipids and lipofuscin in IBA1+ cells. In a larger cohort of SPG11-HSP patients, the ratio of peripheral classical and intermediate monocytes was increased, along with increased serum levels of IL-6 that correlated with disease severity. Stimulation of patient-specific iMGLs with IFNγ led to increased phagocytic activity compared to control iMGL as well as increased upregulation and release of proinflammatory cytokines and chemokines, such as CXCL10. On a molecular basis, we identified increased STAT1 phosphorylation as mechanism connecting IFNγ-mediated immune hyperactivation and SPG11 loss of function. STAT1 expression was increased both in human postmortem brain tissue and in an Spg11-/- mouse model. Application of an STAT1 inhibitor decreased CXCL10 production in SPG11 iMGL and rescued their toxic effect on SPG11 neurons. Our data establish neuroinflammation as a novel disease mechanism in SPG11-HSP patients and constitute the first description of myeloid cell/ microglia activation in human SPG11-HSP. IFNγ/ STAT1-mediated neurotoxic effects of hyperreactive microglia upon SPG11 loss of function indicate that immunomodulation strategies may slow down disease progression.

Efficient and Easy Conversion of Human iPSCs into Functional Induced Microglia-like Cells.

Current protocols converting human induced pluripotent stem cells (iPSCs) into induced microglia-like cells (iMGL) are either dependent on overexpression of transcription factors or require substantial experience in stem-cell technologies. Here, we developed an easy-to-use two-step protocol to convert iPSCs into functional iMGL via: (1) highly efficient differentiation of hematopoietic progenitor cells (HPCs) from iPSCs, and (2) optimized maturation of HPCs to iMGL. A sequential harvesting approach led to an increased HPC yield. The protocol implemented a freezing step, thus allowing HPC biobanking and flexible timing of differentiation into iMGL. Our iMGL responded adequately to the inflammatory stimuli LPS, and iMGL RNAseq analysis matched those of other frequently used protocols. Comparing three different coating modalities, we increased the iMGL yield by culturing on uncoated glass surfaces, thereby retaining differentiation efficiency and functional hallmarks of iMGL. In summary, we provide a high-quality, easy-to-use protocol, rendering generation and functional studies on iMGL an accessible lab resource.

Generation and characterization of an endogenously tagged SPG11-human iPSC line by CRISPR/Cas9 mediated knock-in.

Pathogenic bi-allelic variants in the SPG11 gene result in rare motor neuron disorders such as Hereditary Spastic Paraplegia type 11, Charcot-Marie Tooth, and Juvenile Amyotrophic Lateral Sclerosis-5. The main challenge in SPG11-linked disease research is the lack of antibodies against SPG11 encoded spatacsin. Here, we describe the CRISPR/Cas9 mediated generation and validation of an endogenously tagged SPG11- human iPSC line that contains an HA tag at the C-terminus of SPG11. The line exhibits multi-lineage differentiation potential and holds promise for studying the role of spatacsin and for the elucidation of SPG11-associated pathogenesis. Resource Table.

Disease Modeling of Neuropsychiatric Brain Disorders Using Human Stem Cell-Based Neural Models.

Human pluripotent stem (PS) cells are a relevant platform to model human-specific neurological disorders. In this chapter, we focus on human stem cell models for neuropsychiatric disorders including induced pluripotent stem (iPS) cell-derived neural precursor cells (NPCs), neurons and cerebral organoids. We discuss crucial steps for planning human disease modeling experiments. We introduce the different strategies of human disease modeling including transdifferentiation, human embryonic stem (ES) cell-based models, iPS cell-based models and genome editing options. Analysis of disease-relevant phenotypes is discussed. In more detail, we provide exemplary insight into modeling of the neurodevelopmental defects in autism spectrum disorder (ASD) and the process of neurodegeneration in Alzheimer's disease (AD). Besides monogenic diseases, iPS cell-derived models also generated data from idiopathic and sporadic cases.

Modeling Cell-Cell Interactions in Parkinson's Disease Using Human Stem Cell-Based Models.

Parkinson's disease (PD) is the most frequently occurring movement disorder, with an increasing incidence due to an aging population. For many years, the post-mortem brain was regarded as the gold standard for the analysis of the human pathology of this disease. However, modern stem cell technologies, including the analysis of patient-specific neurons and glial cells, have opened up new avenues for dissecting the pathologic mechanisms of PD. Most data on morphological changes, such as cell death or changes in neurite complexity, or functional deficits were acquired in 2D and few in 3D models. This review will examine the prerequisites for human disease modeling in PD, covering the generation of midbrain neurons, 3D organoid midbrain models, the selection of controls including genetically engineered lines, and the study of cell-cell interactions. We will present major disease phenotypes in human in vitro models of PD, focusing on those phenotypes that have been detected in genetic and sporadic PD models. An additional point covered in this review will be the use of induced pluripotent stem cell (iPSC)-derived technologies to model cell-cell interactions in PD.

Zooming in on Cryopreservation of hiPSCs and Neural Derivatives: A Dual-Center Study Using Adherent Vitrification.

Human induced pluripotent stem cells (hiPSCs) are an important tool for research and regenerative medicine, but their efficient cryopreservation remains a major challenge. The current gold standard is slow-rate freezing of dissociated colonies in suspension, but low recovery rates limit immediate post-thawing applicability. We tested whether ultrafast cooling by adherent vitrification improves post-thawing survival in a selection of hiPSCs and small molecule neural precursor cells (smNPCs) from Parkinson's disease and controls. In a dual-center study, we compared the results by immunocytochemistry (ICC), fluorescence-activated cell sorting analysis, and RNA-sequencing (RNA-seq). Adherent vitrification was achieved in the so-called TWIST substrate, a device combining cultivation, vitrification, storage, and post-thawing cultivation. Adherent vitrification resulted in preserved confluency and significantly higher cell numbers, and viability at day 1 after thawing, while results were not significantly different at day 4 after thawing. RNA-seq and ICC of hiPSCs revealed no change in gene expression and pluripotency markers, indicating that physical damage of slow-rate freezing disrupts cellular membranes. Scanning electron microscopy showed preserved colony integrity by adherent vitrification. Experiments using smNPCs demonstrated that adherent vitrification is also applicable to neural derivatives of hiPSCs. Our data suggest that, compared to the state-of-the-art slow-rate freezing in suspension, adherent vitrification is an improved cryopreservation technique for hiPSCs and derivatives. Stem Cells Translational Medicine 2019;8:247&259.