Conference Agenda

Autosomal Dominant Leukodystrophy (ADLD) is a rare genetic disease associated with white matter loss in the CNS and characterized by autonomic dysfunction and motor impairment. The genetic cause is the presence of three copies of the lamin B1 (LMNB1) gene, which encodes for a structural protein located in the nuclear lamina. Pathogenic mechanisms in ADLD have only initially been explored and a therapy to treat this disease is currently not available.

Based on evidence showing glial pathology in ADLD patients, we generated human glial cells from both ADLD patient- and healthy donor (CTRL)- derived human-induced pluripotent stem cells (hiPSCs), and specifically investigated ADLD astrocytes. Compared to CTRL cells, ADLD astrocytes displayed increased LMNB1 expression, at both RNA and protein level, and morphological and neurochemical cell alterations. Transcriptional profiling of the diseased astrocytes pointed to functional defects in a number of key astrocytic functions, comprising extracellular matrix composition, calcium signaling and mitochondrial metabolism. The analysis further revealed the acquisition of signs of cellular senescence and abnormalities in RNA processing. Importantly, we successfully reduced the elevated levels of LMNB1 protein and reversed the associated cellular abnormalities by using a specific RNA interference technique called Allele SPecific (ASP) RNAi, which selectively silenced the non-duplicated LMNB1 allele.

Our “disease-in-a-dish” platform reveals previously unknown ADLD astroglial dysfunctions, shedding light on their potential contribution to the white matter loss observed in the disease. Moreover, our results provide direct evidence that ASP RNAi can effectively target and alleviate ADLD pathology in human glial cells.

Globoid cell leukodystrophy (GLD) and metachromatic leukodystrophy (MLD) are lysosomal storage diseases caused by deficiency of ß-galactosylceramidase (GALC) and arylsulfatase A (ARSA) enzymes, respectively. Major symptoms are demyelination of the CNS and PNS and neuroinflammation.Over the years, we have used patient-specific human induced pluripotent stem cells (hiPSCs) to model these diseases and explore cell and gene therapy (GT) approaches. We have differentiated hiPSCs into neural stem cell (NSC) populations that display proliferation, long-term self-renewal, and multipotency. By means of RNAseq (bulk and at single-cell resolution) and ChiPseq analysis, we have shown that the hiPSC to neural differentiation is associated with a transcriptional and epigenetic silencing of the pluripotency network and cancer-associated pathways and results in a heterogeneous population that shares similarities with somatic fetal-derived hNSCs. Upon neonatal transplantation, hiPSC-NSCs engraft long-term in murine CNS tissues, disperse widely in several brain regions, and differentiate preferentially into glial cells, suggesting their potential therapeutic role in myelin diseases. Besides being useful for cell therapy approaches hiPSC-NSCs and progeny are excellent tools to model MLD and GLD pathology. Still, the classic 2D neural in vitro models suffer the modest cell maturation of oligodendroglial cells, lack of relevant cell types (i.e. microglia), and lower complexity compared to the brain. Human iPSC-derived brain organoids/spheroids overcome some of these issues, providing a 3D tissue-like framework to better model normal and pathological CNS development and test the efficacy and safety of therapies. Our preliminary data show that 3D spheroids allowing the maturation of both neuronal and oligodendroglial cells that we have generated from patient-specific and isogenic hiPSC clones highlight traits of GLD pathology that were underestimated or not observed at all in 2D models.Overall, these data support the continuous use and refinement of hiPSC-based systems to model and treat leukodystrophies and other demyelinating diseases.

Oligodendrocytes are main targets in demyelinating and dysmyelinating diseases of the central nervous system (CNS), but are also involved in accidental, neurodegenerative and psychiatric disorders. The underlying pathology of these diseases is not fully understood and treatments are still lacking. The recent discovery of the induced pluripotent stem cell (iPSC) technology has opened the possibility to address the biology of human oligodendroglial cells both in the dish and in vivo via engraftment in animal models, and paves the way for the development of treatment for myelin disorders. In this presentation I will address the differences between human and rodent oligodendrocytes, discuss the different sources of human oligodendroglial cells, provide an overview of the different techniques to generate oligodendrocytes from human progenitor or stem cells. I will discuss the anatomical and functional benefit of grafted iPSC-progenitors over their brain counterparts, their use in disease modeling focusing on multiple sclerosis, drug screening, and cell transplantation approaches. Finally, I will talk about the missing gaps that still prevent to study oligodendrocyte biology in the most integrated way, and to translate iPSC-stem cell-based therapy to the clinic.

Autosomal dominant leukodystrophy (ADLD) is a slowly, progressive, genetic, and fatal neurological disorder. The genetic cause of ADLD is Lamin B1 (LMNB1) overexpression due to coding duplications or noncoding deletions at the LMNB1 locus. Lamin B1 is a component of the inner nuclear membrane of cells and although LMNB1 is ubiquitously expressed, it appears that neurons and glial cells are particularly sensible to LMNB1 dosage. Currently, only symptomatic and palliative treatments are available for this fatal disease. Since its discovery, human induced pluripotent stem cell (hiPSC) technology has open to the generation of novel and pathological-relevant in vitro models for Central Nervous System human diseases, for which no appropriate model systems were available. In this work, we describe the reprogramming of peripheral blood mononuclear cell and fibroblast lines derived from ADLD patients carrying different genetic mutations into hiPSCs by Sendai Virus-based method. These hiPSC lines were characterized to assess their pluripotency state by means of qRT-PCR and immunofluorescence assay. Also, embryoid bodies formation assay was used to evaluate their functional pluripotency. In parallel, we set up a procedure for the controlled differentiation of hiPSCs into oligodendrocytes, neurons, and astrocytes. These mature cells were characterized to assess the expression of stage-specific markers by means of qRT-PCR and immunofluorescence assays. In conclusion, patient-derived ADLD hiPSC lines couple to the differentiation protocols that we report represent valuable tools for studies aiming to investigate ADLD-specific alterations at molecular and cellular levels and develop potential target specific drugs.