Publications by authors named "Valentina Fossati"

34 Publications

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

Methods Mol Biol 2021 Apr 10. Epub 2021 Apr 10.

The New York Stem Cell Foundation Research Institute, New York, NY, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/7651_2021_358DOI Listing
April 2021

Human Pluripotent Stem Cell Differentiation to Microglia.

Methods Mol Biol 2021 Mar 28. Epub 2021 Mar 28.

The New York Stem Cell Foundation Research Institute, New York, NY, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/7651_2021_359DOI Listing
March 2021

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

Sci Adv 2021 Jan 6;7(2). Epub 2021 Jan 6.

Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.

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-β neuroinflammation, synaptic signaling, immune activity, mitochondria organization, and myelination. Multiscale network analysis reveals subtype-specific drivers such as , , , , and 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abb5398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787497PMC
January 2021

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

STAR Protoc 2020 Dec 10;1(3):100172. Epub 2020 Nov 10.

The New York Stem Cell Foundation Research Institute, 619 west 54 street, New York, NY 10019, USA.

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 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).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xpro.2020.100172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757411PMC
December 2020

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

Neuron 2021 01 24;109(2):257-272.e14. Epub 2020 Nov 24.

Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2020.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855384PMC
January 2021

Mechanosensitivity of Human Oligodendrocytes.

Front Cell Neurosci 2020 24;14:222. Epub 2020 Jul 24.

Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States.

Oligodendrocytes produce and repair myelin, which is critical for the integrity and function of the central nervous system (CNS). Oligodendrocyte and oligodendrocyte progenitor cell (OPC) biology is modulated by mechanical cues within the magnitudes observed . In some cases, these cues are sufficient to accelerate or inhibit terminal differentiation of murine oligodendrocyte progenitors. However, our understanding of oligodendrocyte lineage mechanobiology has been restricted primarily to animal models to date, due to the inaccessibility and challenges of human oligodendrocyte cell culture. Here, we probe the mechanosensitivity of human oligodendrocyte lineage cells derived from human induced pluripotent stem cells. We target phenotypically distinct stages of the human oligodendrocyte lineage and quantify the effect of substratum stiffness on cell migration and differentiation, within the range documented . We find that human oligodendrocyte lineage cells exhibit mechanosensitive migration and differentiation. Further, we identify two patterns of human donor line-dependent mechanosensitive differentiation. Our findings illustrate the variation among human oligodendrocyte responses, otherwise not captured by animal models, that are important for translational research. Moreover, these findings highlight the importance of studying glia under conditions that better approximate mechanical cues. Despite significant progress in human oligodendrocyte derivation methodology, the extended duration, low yield, and low selectivity of human-induced pluripotent stem cell-derived oligodendrocyte protocols significantly limit the scale-up and implementation of these cells and protocols for and applications. We propose that mechanical modulation, in combination with traditional soluble and insoluble factors, provides a key avenue to address these challenges in cell production and analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2020.00222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7420028PMC
July 2020

CD49f Is a Novel Marker of Functional and Reactive Human iPSC-Derived Astrocytes.

Neuron 2020 08 1;107(3):436-453.e12. Epub 2020 Jun 1.

The New York Stem Cell Foundation Research Institute, New York, NY 10019, USA. Electronic address:

New methods for investigating human astrocytes are urgently needed, given their critical role in the central nervous system. Here we show that CD49f is a novel marker for human astrocytes, expressed in fetal and adult brains from healthy and diseased individuals. CD49f can be used to purify fetal astrocytes and human induced pluripotent stem cell (hiPSC)-derived astrocytes. We provide single-cell and bulk transcriptome analyses of CD49f hiPSC-astrocytes and demonstrate that they perform key astrocytic functions in vitro, including trophic support of neurons, glutamate uptake, and phagocytosis. Notably, CD49f hiPSC-astrocytes respond to inflammatory stimuli, acquiring an A1-like reactive state, in which they display impaired phagocytosis and glutamate uptake and fail to support neuronal maturation. Most importantly, we show that conditioned medium from human reactive A1-like astrocytes is toxic to human and rodent neurons. CD49f hiPSC-astrocytes are thus a valuable resource for investigating human astrocyte function and dysfunction in health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2020.05.014DOI Listing
August 2020

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

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

Brain 2019 09;142(9):2756-2774

Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awz201DOI Listing
September 2019

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

Proc Natl Acad Sci U S A 2019 04 25;116(18):9030-9039. Epub 2019 Mar 25.

Department of Neuroscience, University of Connecticut School of Medicine, Farmington, CT 06030;

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1818348116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500153PMC
April 2019

Cerebrospinal fluid biomarkers link toxic astrogliosis and microglial activation to multiple sclerosis severity.

Mult Scler Relat Disord 2019 Feb 5;28:34-43. Epub 2018 Dec 5.

Neuroimmunological Diseases Section (NDS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 5N248, 10 Center Drive, MSC1444, Bethesda, MD 20892, United States. Electronic address:

Background: Once multiple sclerosis (MS) reaches the progressive stage, immunomodulatory treatments have limited efficacy. This suggests that processes other than activation of innate immunity may at least partially underlie disability progression during late stages of MS. Pathology identified these alternative processes as aberrant activation of astrocytes and microglia, and subsequent degeneration of oligodendrocytes and neurons. However, we mostly lack biomarkers that could measure central nervous system (CNS) cell-specific intrathecal processes in living subjects. This prevents differentiating pathogenic processes from an epiphenomenon. Therefore, we sought to develop biomarkers of CNS cell-specific processes and link them to disability progression in MS.

Methods: In a blinded manner, we measured over 1000 proteins in the cerebrospinal fluid (CSF) of 431 patients with neuroimmunological diseases and healthy volunteers using modified DNA-aptamers (SOMAscan®). We defined CNS cell type-enriched clusters using variable cluster analysis, combined with in vitro modeling. Differences between diagnostic categories were identified in the training cohort (n = 217) and their correlation to disability measures were assessed; results were validated in an independent validation cohort (n = 214).

Results: Astrocyte cluster 8 (MMP7, SERPINA3, GZMA and CLIC1) and microglial cluster 2 (DSG2 and TNFRSF25) were reproducibly elevated in MS and had a significant and reproducible correlation with MS severity suggesting their pathogenic role. In vitro studies demonstrated that proteins of astrocyte cluster 8 are noticeably released upon stimulation with proinflammatory stimuli and overlap with the phenotype of recently described neuro-toxic (A1) astrocytes.

Conclusion: Microglial activation and toxic astrogliosis are associated with MS disease process and may partake in CNS tissue destruction. This hypothesis should be tested in new clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.msard.2018.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411304PMC
February 2019

Oligodendrocyte differentiation of induced pluripotent stem cells derived from subjects with schizophrenias implicate abnormalities in development.

Transl Psychiatry 2018 10 23;8(1):230. Epub 2018 Oct 23.

Harvard Medical School, Boston, MA, USA.

Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F = 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F = 53.6, p < 0.001). A correlation between myelin content in WM in vivo, estimated by magnetization transfer ratio (MTR) and number of O4-positive cells in vitro was also observed across all time points (F = 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p < 0.02). Low production of OPCs may be a contributing mechanism underlying WM reduction in SZ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41398-018-0284-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199264PMC
October 2018

GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease.

Cell Stem Cell 2018 Aug;23(2):239-251.e6

Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA; Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA. Electronic address:

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by mutations in the astrocytic filament gene GFAP. While astrocytes are thought to have important roles in controlling myelination, AxD animal models do not recapitulate critical myelination phenotypes and it is therefore not clear how AxD astrocytes contribute to leukodystrophy. Here, we show that AxD patient iPSC-derived astrocytes recapitulate key features of AxD pathology such as GFAP aggregation. Moreover, AxD astrocytes inhibit proliferation of human iPSC-derived oligodendrocyte progenitor cells (OPCs) in co-culture and reduce their myelination potential. CRISPR/Cas9-based correction of GFAP mutations reversed these phenotypes. Transcriptomic analyses of AxD astrocytes and postmortem brains identified CHI3L1 as a key mediator of AxD astrocyte-induced inhibition of OPC activity. Thus, this iPSC-based model of AxD not only recapitulates patient phenotypes not observed in animal models, but also reveals mechanisms underlying disease pathology and provides a platform for assessing therapeutic interventions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230521PMC
August 2018

Induction of myelinating oligodendrocytes in human cortical spheroids.

Nat Methods 2018 09 25;15(9):700-706. Epub 2018 Jul 25.

Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41592-018-0081-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508550PMC
September 2018

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

Ann Neurol 2017 Nov;82(5):795-812

Neuroimmunological Diseases Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ana.25083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743213PMC
November 2017

Directed Differentiation of Human Pluripotent Stem Cells to Microglia.

Stem Cell Reports 2017 06 18;8(6):1516-1524. Epub 2017 May 18.

The New York Stem Cell Foundation Research Institute, New York, NY 10019, USA.

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2017.04.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470097PMC
June 2017

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

Am J Hum Genet 2017 Apr 30;100(4):617-634. Epub 2017 Mar 30.

Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2017.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384098PMC
April 2017

The silver lining of induced pluripotent stem cell variation.

Stem Cell Investig 2016 6;3:86. Epub 2016 Dec 6.

The New York Stem Cell Foundation Research Institute, New York, NY 10023, USA.

Induced pluripotent stem cells (iPSCs) are being generated using various reprogramming methods and from different cell sources. Hence, a lot of effort has been devoted to evaluating the differences among iPSC lines, in particular with respect to their differentiation capacity. While line-to-line variability should mainly reflect the genetic diversity within the human population, here we review some studies that have brought attention to additional variation caused by genomic and epigenomic alterations. We discuss strategies to evaluate aberrant changes and to minimize technical and culture-induced noise, in order to generate safe cells for clinical applications. We focus on the findings from a recent study, which compared the differentiation capacity of several iPSC lines committed to the hematopoietic lineage and correlated the differential maturation capacity with aberrant DNA methylations. Although iPSC variation represents a challenge for the field, we embrace the authors' perspective that iPSC variations should be used to our advantage for predicting and selecting the best performing iPSC lines, depending on the desired application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21037/sci.2016.11.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182208PMC
December 2016

A viral strategy for targeting and manipulating interneurons across vertebrate species.

Nat Neurosci 2016 12 31;19(12):1743-1749. Epub 2016 Oct 31.

NYU Neuroscience Institute, New York University Langone Medical Center, New York, New York, USA.

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nn.4430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348112PMC
December 2016

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

Int J Mol Sci 2016 Apr 22;17(4). Epub 2016 Apr 22.

The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms17040614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849063PMC
April 2016

Characterization of molecular and cellular phenotypes associated with a heterozygous deletion using patient-derived hiPSC neural cells.

NPJ Schizophr 2015 Jun;1

Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029.

Neurodevelopmental disorders, such as autism spectrum disorders (ASD) and schizophrenia (SZ), are complex disorders with a high degree of heritability. Genetic studies have identified several candidate genes associated with these disorders, including contactin-associated protein-like 2 (). Traditionally, in animal models or , the function of has been studied by genetic deletion or transcriptional knockdown, which reduce the expression of the entire gene; however, it remains unclear whether the mutations identified in clinical settings are sufficient to alter expression in human neurons. Here, using human induced pluripotent stem cells (hiPSCs) derived from two individuals with a large (289kb) and heterozygous deletion in (affecting exons 14-15) and discordant clinical outcomes, we have characterized expression patterns in hiPSC neural progenitor cells (NPCs), two independent populations of hiPSC-derived neurons and hiPSC-derived oligodendrocyte precursor cells (OPCs). First, we observed exon-specific changes in expression in both carriers; although the expression of exons 14-15 is significantly decreased, the expression of other exons is upregulated. Second, we observed significant differences in patterns of allele-specific expression in carriers that were consistent with clinical outcome. Third, we observed a robust neural migration phenotype that correlated with diagnosis and exon- and allele-specific expression patterns, but not with genotype. In all, our data highlight the importance of considering the nature, location and regulation of mutated alleles when attempting to connect GWAS studies to gene function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789165PMC
http://dx.doi.org/10.1038/npjschz.2015.19DOI Listing
June 2015

Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells.

Nat Protoc 2015 Aug 2;10(8):1143-54. Epub 2015 Jul 2.

The New York Stem Cell Foundation Research Institute, New York, New York, USA.

In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor-rich medium. Within 8 d, PSCs differentiate into paired box 6-positive (PAX6(+)) neural stem cells, which give rise to OLIG2(+) progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2(+)) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4(+)) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4(+) OPCs can be isolated by cell sorting for myelination studies, or they can be terminally differentiated to myelin basic protein-positive (MBP(+)) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4(+) cells after only 55 d of culture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nprot.2015.075DOI Listing
August 2015

Generating induced pluripotent stem cells for multiple sclerosis therapy.

Regen Med 2014 ;9(6):709-11

The New York Stem Cell Foundation Research Institute, NY 10032, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/rme.14.63DOI Listing
July 2015

Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells.

Stem Cell Reports 2014 Aug 24;3(2):250-9. Epub 2014 Jul 24.

The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA. Electronic address:

Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology that affects the CNS. While current therapies are primarily directed against the immune system, the new challenge is to address progressive MS with remyelinating and neuroprotective strategies. Here, we develop a highly reproducible protocol to efficiently derive oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from induced pluripotent stem cells (iPSCs). Key elements of our protocol include adherent cultures, dual SMAD inhibition, and addition of retinoids from the beginning of differentiation, which lead to increased yields of OLIG2 progenitors and high numbers of OPCs within 75 days. Furthermore, we show the generation of viral and integration-free iPSCs from primary progressive MS (PPMS) patients and their efficient differentiation to oligodendrocytes. PPMS OPCs are functional, as demonstrated by in vivo myelination in the shiverer mouse. These results provide encouraging advances toward the development of autologous cell therapies using iPSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2014.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176529PMC
August 2014

How big is the myelinating orchestra? Cellular diversity within the oligodendrocyte lineage: facts and hypotheses.

Front Cell Neurosci 2014 28;8:201. Epub 2014 Jul 28.

The New York Stem Cell Foundation New York, NY, USA.

Since monumental studies from scientists like His, Ramón y Cajal, Lorente de Nó and many others have put down roots for modern neuroscience, the scientific community has spent a considerable amount of time, and money, investigating any possible aspect of the evolution, development and function of neurons. Today, the complexity and diversity of myriads of neuronal populations, and their progenitors, is still focus of extensive studies in hundreds of laboratories around the world. However, our prevalent neuron-centric perspective has dampened the efforts in understanding glial cells, even though their active participation in the brain physiology and pathophysiology has been increasingly recognized over the years. Among all glial cells of the central nervous system (CNS), oligodendrocytes (OLs) are a particularly specialized type of cells that provide fundamental support to neuronal activity by producing the myelin sheath. Despite their functional relevance, the developmental mechanisms regulating the generation of OLs are still poorly understood. In particular, it is still not known whether these cells share the same degree of heterogeneity of their neuronal companions and whether multiple subtypes exist within the lineage. Here, we will review and discuss current knowledge about OL development and function in the brain and spinal cord. We will try to address some specific questions: do multiple OL subtypes exist in the CNS? What is the evidence for their existence and those against them? What are the functional features that define an oligodendrocyte? We will end our journey by reviewing recent advances in human pluripotent stem cell differentiation towards OLs. This exciting field is still at its earliest days, but it is quickly evolving with improved protocols to generate functional OLs from different spatial origins. As stem cells constitute now an unprecedented source of human OLs, we believe that they will become an increasingly valuable tool for deciphering the complexity of human OL identity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2014.00201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112809PMC
August 2014

The sixth annual translational stem cell research conference of the New York Stem Cell Foundation.

Ann N Y Acad Sci 2012 May 28;1255:16-29. Epub 2012 Mar 28.

The New York Stem Cell Foundation, New York, New York 10023, USA.

The New York Stem Cell Foundation's "Sixth Annual Translational Stem Cell Research Conference" convened on October 11-12, 2011 at the Rockefeller University in New York City. Over 450 scientists, patient advocates, and stem cell research supporters from 14 countries registered for the conference. In addition to poster and platform presentations, the conference featured panels entitled "Road to the Clinic" and "The Future of Regenerative Medicine."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1749-6632.2012.06481.xDOI Listing
May 2012

The New York Stem Cell Foundation: Fifth Annual Translational Stem Cell Research Conference.

Ann N Y Acad Sci 2011 May;1226:1-13

The New York Stem Cell Foundation, New York, New York 10023, USA.

The New York Stem Cell Foundation's "Fifth Annual Translational Stem Cell Research Conference" convened on October 12-13, 2010 at the Rockefeller University in New York City. The conference attracted over 400 scientists, patient advocates, and stem cell research supporters from 16 countries. In addition to poster and platform presentations, the conference featured panels entitled "Road to the Clinic" and "Regulatory Roadblocks."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1749-6632.2011.06038.xDOI Listing
May 2011

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

J Immunol 2010 Feb 28;184(3):1251-60. Epub 2009 Dec 28.

Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.0901922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2809811PMC
February 2010