Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNß and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

Cellular senescence in progenitor cells contributes to diminished remyelination potential in progressive multiple sclerosis.

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

Induction of myelinating oligodendrocytes in human cortical spheroids.

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes.

Pelizaeus-Merzbacher disease (PMD) is a pediatric disease of myelin in the central nervous system and manifests with a wide spectrum of clinical severities. Although PMD is a rare monogenic disease, hundreds of mutations in the X-linked myelin gene proteolipid protein 1 (PLP1) have been identified in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hiPSC-derived oligodendrocytes from 12 individuals with mutations spanning the genetic and clinical diversity of PMD-including point mutations and duplication, triplication, and deletion of PLP1-and developed an in vitro platform for molecular and cellular characterization of all 12 mutations simultaneously. We identified individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted testing of small-molecule modulators of the endoplasmic reticulum stress response, which improved both morphologic and myelination defects. Collectively, these data provide insights into the pathogeneses of a variety of PLP1 mutations and suggest that disparate etiologies of PMD could require specific treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin disorder.

A metabolic perspective on CSF-mediated neurodegeneration in multiple sclerosis.

Multiple sclerosis is an autoimmune demyelinating disorder of the CNS, characterized by inflammatory lesions and an underlying neurodegenerative process, which is more prominent in patients with progressive disease course. It has been proposed that mitochondrial dysfunction underlies neuronal damage, the precise mechanism by which this occurs remains uncertain. To investigate potential mechanisms of neurodegeneration, we conducted a functional screening of mitochondria in neurons exposed to the CSF of multiple sclerosis patients with a relapsing remitting (n = 15) or a progressive (secondary, n = 15 or primary, n = 14) disease course. Live-imaging of CSF-treated neurons, using a fluorescent mitochondrial tracer, identified mitochondrial elongation as a unique effect induced by the CSF from progressive patients. These morphological changes were associated with decreased activity of mitochondrial complexes I, III and IV and correlated with axonal damage. The effect of CSF treatment on the morphology of mitochondria was characterized by phosphorylation of serine 637 on the dynamin-related protein DRP1, a post-translational modification responsible for unopposed mitochondrial fusion in response to low glucose conditions. The effect of neuronal treatment with CSF from progressive patients was heat stable, thereby prompting us to conduct an unbiased exploratory lipidomic study that identified specific ceramide species as differentially abundant in the CSF of progressive patients compared to relapsing remitting multiple sclerosis. Treatment of neurons with medium supplemented with ceramides, induced a time-dependent increase of the transcripts levels of specific glucose and lactate transporters, which functionally resulted in progressively increased glucose uptake from the medium. Thus ceramide levels in the CSF of patients with progressive multiple sclerosis not only impaired mitochondrial respiration but also decreased the bioavailability of glucose by increasing its uptake. Importantly the neurotoxic effect of CSF treatment could be rescued by exogenous supplementation with glucose or lactate, presumably to compensate the inefficient fuel utilization. Together these data suggest a condition of 'virtual hypoglycosis' induced by the CSF of progressive patients in cultured neurons and suggest a critical temporal window of intervention for the rescue of the metabolic impairment of neuronal bioenergetics underlying neurodegeneration in multiple sclerosis patients.

Molecular-based diagnosis of multiple sclerosis and its progressive stage.

OBJECTIVE: Biomarkers aid diagnosis, allow inexpensive screening of therapies, and guide selection of patient-specific therapeutic regimens in most internal medicine disciplines. In contrast, neurology lacks validated measurements of the physiological status, or dysfunction(s) of cells of the central nervous system (CNS). Accordingly, patients with chronic neurological diseases are often treated with a single disease-modifying therapy without understanding patient-specific drivers of disability. Therefore, using multiple sclerosis (MS) as an example of a complex polygenic neurological disease, we sought to determine whether cerebrospinal fluid (CSF) biomarkers are intraindividually stable, cell type-, disease- and/or process-specific, and responsive to therapeutic intervention. METHODS: We used statistical learning in a modeling cohort (n = 225) to develop diagnostic classifiers from DNA-aptamer-based measurements of 1,128 CSF proteins. An independent validation cohort (n = 85) assessed the reliability of derived classifiers. The biological interpretation resulted from in vitro modeling of primary or stem cell-derived human CNS cells and cell lines. RESULTS: The classifier that differentiates MS from CNS diseases that mimic MS clinically, pathophysiologically, and on imaging achieved a validated area under the receiver operating characteristic curve (AUROC) of 0.98, whereas the classifier that differentiates relapsing-remitting from progressive MS achieved a validated AUROC of 0.91. No classifiers could differentiate primary progressive from secondary progressive MS better than random guessing. Treatment-induced changes in biomarkers greatly exceeded intraindividual and technical variabilities of the assay. INTERPRETATION: CNS biological processes reflected by CSF biomarkers are robust, stable, disease specific, or even disease stage specific. This opens opportunities for broad utilization of CSF biomarkers in drug development and precision medicine for CNS disorders. Ann Neurol 2017;82:795-812.

Epigenetic Modulation of Human Induced Pluripotent Stem Cell Differentiation to Oligodendrocytes.

Pluripotent stem cells provide an invaluable tool for generating human, disease-relevant cells. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system, characterized by myelin damage. Oligodendrocytes are the myelinating cells of the central nervous system (CNS); they differentiate from progenitor cells, and their membranes ensheath axons, providing trophic support and allowing fast conduction velocity. The current understanding of oligodendrocyte biology was founded by rodent studies, where the establishment of repressive epigenetic marks on histone proteins, followed by activation of myelin genes, leads to lineage progression. To assess whether this epigenetic regulation is conserved across species, we differentiated human embryonic and induced pluripotent stem cells to oligodendrocytes and asked whether similar histone marks and relative enzymatic activities could be detected. The transcriptional levels of enzymes responsible for methylation and acetylation of histone marks were analyzed during oligodendrocyte differentiation, and the post-translational modifications on histones were detected using immunofluorescence. These studies showed that also in human cells, differentiation along the oligodendrocyte lineage is characterized by the acquisition of multiple repressive histone marks, including deacetylation of lysine residues on histone H3 and trimethylation of residues K9 and K27. These data suggest that the epigenetic modulation of oligodendrocyte identity is highly conserved across species.

Establishing Neural Organoid Cultures for Investigating the Effects of Microgravity in Low-Earth Orbit (LEO).

Recent findings from studies involving astronauts and animal models indicate that microgravity increases immune cell activity and potentially alters the white and gray matter of the central nervous system (CNS). To further investigate the impact of microgravity on CNS cells, we established cultures of three-dimensional neural organoids containing isogenic microglia, the brain's resident immune cells, and sent them onboard the International Space Station. When using induced pluripotent stem cell (iPSC) lines from individuals affected by neuroinflammatory and neurodegenerative diseases such as multiple sclerosis (MS) and Parkinson's disease (PD), these cultures can provide novel insights into pathogenic pathways that may be exacerbated by microgravity. We have devised a cryovial culture strategy that enables organoids to be maintained through space travel and onboard the International Space Station (ISS) without the need for medium or carbon dioxide exchange. Here, we provide a comprehensive description of all the steps involved: generating various types of neural organoids, establishing long-term cultures, arranging plans for shipment to the Kennedy Space Center (KSC), and ultimately preparing organoids for launch into low-Earth orbit (LEO) and return to Earth for post-flight analyses.

New records of non-indigenous species from the eastern Mediterranean Sea (Crustacea, Mollusca), with a revision of genus Isognomon (Mollusca: Bivalvia).

We report new data on non-indigenous invertebrates from the Mediterranean Sea (four ostracods and 20 molluscs), including five new records for the basin: the ostracods Neomonoceratina iniqua, Neomonoceratina aff. mediterranea, Neomonoceratina cf. entomon, Loxoconcha cf. gisellae (Arthropoda: Crustacea)-the first records of non-indigenous ostracods in the Mediterranean-and the bivalve Striarca aff. symmetrica (Mollusca). Additionally, we report for the first time Electroma vexillum from Israel, and Euthymella colzumensis, Joculator problematicus, Hemiliostraca clandestina, Pyrgulina nana, Pyrgulina microtuber, Turbonilla cangeyrani, Musculus aff. viridulus and Isognomon bicolor from Cyprus. We also report the second record of Fossarus sp. and of Cerithiopsis sp. cf. pulvis in the Mediterranean Sea, the first live collected specimens of Oscilla galilae from Cyprus and the northernmost record of Gari pallida in Israel (and the Mediterranean). Moreover, we report the earliest records of Rugalucina angela, Ervilia scaliola and Alveinus miliaceus in the Mediterranean Sea, backdating their first occurrence in the basin by 3, 5 and 7 years, respectively. We provide new data on the presence of Spondylus nicobaricus and Nudiscintilla aff. glabra in Israel. Finally, yet importantly, we use both morphological and molecular approaches to revise the systematics of the non-indigenous genus Isognomon in the Mediterranean Sea, showing that two species currently co-occur in the basin: the Caribbean I. bicolor, distributed in the central and eastern Mediterranean, and the Indo-Pacific I. aff. legumen, at present reported only from the eastern Mediterranean and whose identity requires a more in-depth taxonomic study. Our work shows the need of taxonomic expertise and investigation, the necessity to avoid the unfounded sense of confidence given by names in closed nomenclature when the NIS belong to taxa that have not enjoyed ample taxonomic work, and the necessity to continue collecting samples-rather than relying on visual censuses and bio-blitzes-to enable accurate detection of non-indigenous species.

Longitudinal scRNA-seq analysis in mouse and human informs optimization of rapid mouse astrocyte differentiation protocols.

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence during the development of the brain and spinal cord. Here we used single-cell/single-nucleus RNA sequencing (scRNA-seq/snRNA-seq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human-induced pluripotent stem cells. We computationally identify candidate genes involved in the fate specification of glia in both species and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our transcriptomic data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multi-omic, dual single-nuclei (sn)RNA-seq/snATAC-seq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets will enable future optimization of glial differentiation protocols and provide insight into human glial differentiation.

Patient iPSC models reveal glia-intrinsic phenotypes in multiple sclerosis.

Multiple sclerosis (MS) is considered an inflammatory and neurodegenerative disease of the central nervous system, typically resulting in significant neurological disability that worsens over time. While considerable progress has been made in defining the immune system's role in MS pathophysiology, the contribution of intrinsic CNS-cell dysfunction remains unclear. Here, we generated the largest reported collection of iPSC lines from people with MS spanning diverse clinical subtypes and differentiated them into glia-enriched cultures. Using single-cell transcriptomic profiling, we observed several distinguishing characteristics of MS cultures pointing to glia-intrinsic disease mechanisms. We found that iPSC-derived cultures from people with primary progressive MS contained fewer oligodendrocytes. Moreover, iPSC-oligodendrocyte lineage cells and astrocytes from people with MS showed increased expression of immune and inflammatory genes that match those of glial cells from MS postmortem brains. Thus, iPSC-derived MS models provide a unique platform for dissecting glial contributions to disease phenotypes independent of the peripheral immune system and identify potential glia-specific targets for therapeutic intervention.

Progenitor cell origin plays a role in fate choices of mature B cells.

B cells, the Ab-producing cells of the immune system, develop from hematopoietic stem cells (HSCs) through well-defined stages during which Ig genes are rearranged to generate a clonal BCR. Signaling through the BCR plays a role in the subsequent cell fate decisions leading to the generation of three distinct types of B cells: B1, marginal zone, and follicular B cells. Common lymphoid progenitors (CLPs) are descended from HSCs, and although recent observations suggest that CLPs may not be physiological T cell precursors, it is generally accepted that CLPs are obligate progenitors for B cells. In addition, a CLP-like progenitor of unknown significance that lacks expression of c-kit (kit(-)CLP) was recently identified in the mouse model. In this study, we show that CLPs, kit(-)CLPs and a population within the lin(-)Sca1(+)kit(+)flt3(-) HSC compartment generate mature B cell types in different proportions: CLPs and kit(-)CLPs show a stronger marginal zone/follicular ratio than lin(-)Sca1(+)kit(+)flt3(-) cells, whereas kit(-)CLPs show a stronger B1 bias than any other progenitor population. Furthermore, expression of Sca1 on B cells depends on their progenitor origin as B cells derived from CLPs and kit(-)CLPs express more Sca1 than those derived from lin(-)Sca1(+)kit(+)flt3(-) cells. These observations indicate a role for progenitor origin in B cell fate choices and suggest the existence of CLP-independent B cell development.

Human Induced Pluripotent Stem Cell (iPSC) Handling Protocols: Maintenance, Expansion, and Cryopreservation.

Human induced pluripotent stem cells (iPSCs) have emerged as an invaluable resource for basic research, disease modeling, and drug discovery over recent years. Given the numerous advantages of iPSCs over alternative models-including their human origin, their ability to be differentiated into almost any cell type, and the therapeutic potential of patient-specific iPSCs in personalized medicine-many labs are now considering iPSC models for their studies. As the quality of the starting population of iPSCs is a key determinant in the success of any one of these applications, it is crucial to adhere to best practices in iPSC culture. In the following protocol, we offer a comprehensive guide to the culture, cryopreservation, and quality control methods required for the establishment and maintenance of high-quality iPSC cultures.

Human Pluripotent Stem Cell Differentiation to Microglia.

Microglia, the immune cells of the central nervous system (CNS), play critical roles in CNS homeostasis and disease. Mounting evidence has linked aberrant microglial functions to neurodevelopment, neuroinflammatory and neurodegenerative diseases, underlining the need for novel models to investigate human microglia biology. Here we describe a protocol for generating in vitro patient-specific microglia progenitors and microglia-like cells from induced pluripotent stem cells (iPSCs). Our protocol generates microglia progenitor cells in approximately 35 days, which then can further mature into microglia-like cells within two additional weeks. Microglia differentiation is driven by specific growth factors and cytokines in serum-free conditions, resulting in mesodermal progenitors that grow in a monolayer which releases free-floating microglia progenitors. Isolated progenitors can be used in co-culture systems with other neuronal cells, xenotransplanted to generate chimeric mouse models, or further differentiated into adherent microglia-like cells for functional studies.

Reactive Astrocytes Derived From Human Induced Pluripotent Stem Cells Suppress Oligodendrocyte Precursor Cell Differentiation.

Astrocytes are instrumental in maintaining central nervous system (CNS) homeostasis and responding to injury. A major limitation of studying neurodegenerative diseases like multiple sclerosis (MS) is lack of human pathological specimens obtained during the acute stages, thereby relegating research to post-mortem specimens obtained years after the initiation of pathology. Rodent reactive astrocytes have been shown to be cytotoxic to neurons and oligodendrocytes but may differ from human cells, especially in diseases with genetic susceptibility. Herein, we purified human CD49f+ astrocytes from induced pluripotent stem cells derived from individual patient and control peripheral leukocytes. We compared TNF and IL1α stimulated human reactive astrocytes from seven persons with MS and six non-MS controls and show their transcriptomes are remarkably similar to those described in rodents. The functional effect of astrocyte conditioned media (ACM) was examined in a human oligodendrocyte precursor cell (OPC) line differentiation assay. ACM was not cytotoxic to the OPCs but robustly inhibited the myelin basic protein (MBP) reporter. No differences were seen between MS and control stimulated astrocytes at either the transcript level or in ACM mediated OPC suppression assays. We next used RNAseq to interrogate differentially expressed genes in the OPC lines that had suppressed differentiation from the human ACM. Remarkably, not only was OPC differentiation and myelin gene expression suppressed, but we observed induction of several immune pathways in OPCs exposed to the ACM. These data support the notion that reactive astrocytes can inhibit OPC differentiation thereby limiting their remyelination capacity, and that OPCs take on an immune profile in the context of inflammatory cues.

Proteomic Alterations and Novel Markers of Neurotoxic Reactive Astrocytes in Human Induced Pluripotent Stem Cell Models.

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP+ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.

Molecular subtyping of Alzheimer's disease using RNA sequencing data reveals novel mechanisms and targets.

Alzheimer's disease (AD), the most common form of dementia, is recognized as a heterogeneous disease with diverse pathophysiologic mechanisms. In this study, we interrogate the molecular heterogeneity of AD by analyzing 1543 transcriptomes across five brain regions in two AD cohorts using an integrative network approach. We identify three major molecular subtypes of AD corresponding to different combinations of multiple dysregulated pathways, such as susceptibility to tau-mediated neurodegeneration, amyloid-ß neuroinflammation, synaptic signaling, immune activity, mitochondria organization, and myelination. Multiscale network analysis reveals subtype-specific drivers such as GABRB2, LRP10, MSN, PLP1, and ATP6V1A We further demonstrate that variations between existing AD mouse models recapitulate a certain degree of subtype heterogeneity, which may partially explain why a vast majority of drugs that succeeded in specific mouse models do not align with generalized human trials across all AD subtypes. Therefore, subtyping patients with AD is a critical step toward precision medicine for this devastating disease.

Transformative Network Modeling of Multi-omics Data Reveals Detailed Circuits, Key Regulators, and Potential Therapeutics for Alzheimer's Disease.

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD.

Lin-Sca1+kit- bone marrow cells contain early lymphoid-committed precursors that are distinct from common lymphoid progenitors.

The significance of a population in mouse bone marrow of lineage-negative (Lin(-)), Sca1-positive, c-kit-negative (LSK(-)) cells, which is reported to be devoid of long-term repopulation capacity or myeloid potential, is unknown. In this study, we show that the LSK(-) population is composed of several subsets defined by the expression of flt3, CD25, and IL-7Ralpha. The first subset was CD25(-) and more than 90% expressed either flt3, IL-7Ralpha, or both. The CD25(-)LSK(-) population had T cell, B cell, and NK cell potential in vivo, and most of this activity was localized in the flt3(+) subset, irrespective of the expression of IL-7Ralpha. Although lymphoid potential of flt3(+)LSK(-) cells in vivo was 3-fold lower than that of lin(-)Sca1(low)kit(low)IL7Ralpha(+) common lymphoid progenitors (CLPs), their cloning efficiency in vitro was 10-fold lower than that of CLPs. Furthermore, although the myeloid potential of flt3(+)LSK(-) cells was 10-fold lower than that of CLPs in the absence of M-CSF, the relative myeloid potential of both populations was similar in its presence. These observations suggest differential growth factor requirements of both populations. The second subset of LSK(-) cells was homogeneously CD25(+)flt3(-)IL7Ralpha(+) and could be generated from both CD25(-)LSK(-) cells and from CLPs, but did not engraft in immunodeficient Rag1(-/-) or Rag1(-/-)gamma(c)(-/-) hosts. This population, of which the significance is unclear, was increased in Rag1(-/-) mice and in old mice. Thus, the LSK(-) population is phenotypically and functionally heterogeneous and contains early lymphoid-committed precursors. Our findings imply that the early stages of lymphoid commitment are more complex than was thus far assumed.

Isolation of Human CD49f+ Astrocytes and In Vitro iPSC-Based Neurotoxicity Assays.

Given the critical roles of astrocytes in neuroinflammation and neurological diseases, models for studying human astrocyte biology are in increasing demand. Here, we present a protocol to isolate human astrocytes from induced pluripotent stem cell (iPSC)-based cultures, neural organoids, and primary tissue, using the surface marker CD49f. Moreover, we provide protocols for in vitro co-cultures of human iPSC-derived neurons and astrocytes, as well as for neurotoxicity assays that expose neurons to conditioned media from reactive astrocytes. For complete details on the use and execution of this protocol, please refer to Barbar et al. (2020).