Valentina Fossati

Efficient Generation of Myelinating Oligodendrocytes from Primary Progressive Multiple Sclerosis Patients by Induced Pluripotent Stem Cells

Summary 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.

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

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.

A viral strategy for targeting and manipulating interneurons across vertebrate species

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.

Directed Differentiation of Human Pluripotent Stem Cells to Microglia

Summary 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 Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

Characterization of molecular and cellular phenotypes associated with a heterozygous CNTNAP2 deletion using patient-derived hiPSC neural cells

Neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia, 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 (CNTNAP2). Traditionally, in animal models or in vitro, CNTNAP2 has been studied by genetic deletion or transcriptional knockdown, which reduces the expression of the entire gene; however, it remains unclear whether the mutations identified in clinical settings are sufficient to alter CNTNAP2 expression in human neurons. Here, using human induced pluripotent stem cells (hiPSCs) derived from two individuals with a large (289 kb) heterozygous deletion in CNTNAP2 (affecting exons 14–15) and discordant clinical outcomes, we have characterized CNTNAP2 expression patterns in hiPSC neural progenitor cells, two independent populations of hiPSC-derived neurons and hiPSC-derived oligodendrocyte precursor cells. First, we observed exon-specific changes in CNTNAP2 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 CNTNAP2 carriers that were consistent with the clinical outcome. Third, we observed a robust neural migration phenotype that correlated with diagnosis and exon- and allele-specific CNTNAP2 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 genome wide association studies to gene function.

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

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.

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.

Generating induced pluripotent stem cells for multiple sclerosis therapy.

Over 150 years of research in multiple sclerosis (MS) culminated, during the last two decades, in the approval of several disease-modifying drugs, from the first selfinjectable immunomodulators, to the latest oral compounds and in several promising clinical trials testing the efficacy of monoclonal antibodies against B or T cells, and hematopoietic and mesenchymal stem cells transplantation. All those strategies have more or less succeeded at preventing damage from an immune system gone astray. The next challenge in the MS field is to develop neuroprotective and re-myelinating paradigms. In the meantime, the scientific community has witnessed a stem cell revolution, with the derivation of human embryonic stem cells, followed – less than 10 years later – by the groundbreaking discovery that adult cells could be reprogrammed to revert to an undifferentiated ‘embryonic-like’ stage and become induced pluripotent stem cells (iPSCs). Today, iPSCs play a central role in an increasing number of studies focusing on both disease modeling and cell-replacement therapies including those for neurological, muscular, ocular, cardiac, hematological, metabolic and skin disorders [1]. Needless to say, the question arises of whether iPSCs can effectively impact MS research, supporting the emerging routes of investigation, focusing on neurodegeneration/neuroprotection and de-myelination/re-myelination. Chronic neurodegeneration, etiology of which is mostly unknown, distinguishes patients that switch to a secondary progressive phase over time and patients that are affected by the most severe primary progressive form of MS. The axonal dysfunction has been traditionally seen as the consequence of an autoimmune-mediated demyelination. However increasing evidence is challenging this concept and suggesting that neurodegeneration is an independent phenomenon, occurring from the onset and ultimately leading to an accumulation of physical and cognitive disabilities [2,3]. Understanding the pathogenesis of neurodegeneration in order to develop neuroprotective strategies for progressive patients has become an urgent priority. Thus far, studies conducted with primary focus in this direction have been hampered by the limited access to histopathological specimens from the patients and by the lack of an appropriate animal model that would recapitulate the spontaneous onset of the human disease. iPSCs offer the unprecedented opportunity to generate patient-derived cells in substantial quantity; protocols to generate different type of neurons have been developed at remarkable pace and are constantly improving [4,5]; and most importantly, many independent studies have now proven that iPSC-derived cells can recapitulate in vitro disease-relevant phenotypes and provide crucial information regarding the pathogenesis of the disease [6–8]. Extremely encouraging is the fact that even a complex genetic psychiatric disorder such as schizophrenia has been modeled through iPSCs [7]. The simplicity of cell cultures is certainly a limitation for the analysis of extrinsic factors involved in the development of a disease. On the other hand, they can provide a controlled Generating induced pluripotent stem cells for multiple sclerosis therapy

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.A method for generating cortical spheroids from human pluripotent stem cells produces maturing oligodendrocytes that can myelinate axons and model myelin disease and drug effects.

Molecular-based diagnosis of Multiple Sclerosis and its progressive stage

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.