Marc Ehrlich

Human stem cell models of neurodegeneration: a novel approach to study mechanisms of disease development

The number of patients with neurodegenerative diseases is increasing significantly worldwide. Thus, intense research is being pursued to uncover mechanisms of disease development in an effort to identify molecular targets for therapeutic intervention. Analysis of postmortem tissue from patients has yielded important histological and biochemical markers of disease progression. However, this approach is inherently limited because it is not possible to study patient neurons prior to degeneration. As such, transgenic and knockout models of neurodegenerative diseases are commonly employed. While these animal models have yielded important insights into some molecular mechanisms of disease development, they do not provide the opportunity to study mechanisms of neurodegeneration in human neurons at risk and thus, it is often difficult or even impossible to replicate human pathogenesis with this approach. The generation of patient-specific induced pluripotent stem (iPS) cells offers a unique opportunity to overcome these obstacles. By expanding and differentiating iPS cells, it is possible to generate large numbers of functional neurons in vitro, which can then be used to study the disease of the donating patient. Here, we provide an overview of human stem cell models of neurodegeneration using iPS cells from patients with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington’s disease, spinal muscular atrophy and other neurodegenerative diseases. In addition, we describe how further refinements of reprogramming technology resulted in the generation of patient-specific induced neurons, which have also been used to model neurodegenerative changes in vitro.

Origin-Dependent Neural Cell Identities in Differentiated Human iPSCs In Vitro and after Transplantation into the Mouse Brain

The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors

Significance Understanding of myelin diseases and development of new treatment options are at least partly hampered by the limited availability of human oligodendrocytes. Induced pluripotent stem cells (iPSC) may be an ideal tool to circumvent this problem; however, rapid and efficient protocols to generate oligodendrocytes from human iPSC are currently lacking. The induction of the transcription factors SOX10, OLIG2, and NKX6.2 in iPSC-derived neural progenitors accelerates oligodendroglial differentiation significantly resulting in up to 70% of O4+ oligodendrocytes within 28 d. These oligodendrocytes myelinate the CNS during development and after demyelination, and are suitable for pharmacological screens and disease modeling. The strategy presented herein will markedly facilitate the studying of human myelin diseases and the development of screening platforms for drug discovery. Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shi Rag−/− mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

Astrocyte pathology in a human neural stem cell model of frontotemporal dementia caused by mutant TAU protein

Astroglial pathology is seen in various neurodegenerative diseases including frontotemporal dementia (FTD), which can be caused by mutations in the gene encoding the microtubule-associated protein TAU (MAPT). Here, we applied a stem cell model of FTD to examine if FTD astrocytes carry an intrinsic propensity to degeneration and to determine if they can induce non-cell-autonomous effects in neighboring neurons. We utilized CRISPR/Cas9 genome editing in human induced pluripotent stem (iPS) cell-derived neural progenitor cells (NPCs) to repair the FTD-associated N279K MAPT mutation. While astrocytic differentiation was not impaired in FTD NPCs derived from one patient carrying the N279K MAPT mutation, FTD astrocytes appeared larger, expressed increased levels of 4R-TAU isoforms, demonstrated increased vulnerability to oxidative stress and elevated protein ubiquitination and exhibited disease-associated changes in transcriptome profiles when compared to astrocytes derived from one control individual and to the isogenic control. Interestingly, co-culture experiments with FTD astrocytes revealed increased oxidative stress and robust changes in whole genome expression in previously healthy neurons. Our study highlights the utility of iPS cell-derived NPCs to elucidate the role of astrocytes in the pathogenesis of FTD.

Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain.

Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.

Recovery from Toxic-Induced Demyelination Does Not Require the NG2 Proteoglycan

NG2 cells are defined as CNS cells expressing chondroitin sulfate proteoglycan nerve/glia antigen. The vast majority of NG2-positive cells also express platelet-derived growth factor receptor alpha (PDGFRα) and are oligodendroglial progenitors (OPC). In addition a subpopulation of pericytes expresses NG2, but is positive for PDGF receptor beta (PDGFRβ) [1]. NG2-positive OPC comprise approximately 5% of the cells in the CNS where they are evenly distributed in grey and white matter [2, 3]. NG2-positive OPC form synapses with neurons [4–6] and react to brain injury with proliferation, as has been shown in several animal models as well as in human demyelinating and degenerative diseases [7–9]. In vitro, NG2 positive cells can give rise to oligodendrocytes, astrocytes and occasional neurons depending on cell culture conditions [10–12]. In vivo, NG2 cells generate mostly oligodendrocytes as well as small populations of astrocytes as has been demonstrated in fate mapping studies [9, 13–16]. The developmental fate switch from the oligodendroglial into the astrocytic lineage is regulated by Olig2 [17]. A large percentage of NG2 positive cells persists as a self-renewing population in the adult CNS [18–21]. Although NG2 has been extensively used as a marker for OPC, relatively little is known about the functional role of the NG2 proteoglycan. NG2 consists of a small intracellular and a large extracellular domain. The extracellular domain is cleaved by proteases such as ADAM 10 in an activity-dependent fashion, which regulates glutamate signalling at nearby neurons. The NG2 extracellular domain binds receptors, growth factors, extracellular matrix components and proteases (for review see [22, 23]). Lack of NG2 expression in NG2 deficient (NG2-/-) mice or pharmacological inhibition of NG2 ectodomain shedding in wild type OPC results in NMDA and AMPA receptor-dependent reduction of neuronal current amplitudes and an altered behaviour of NG2-/- mice in tests measuring sensorimotor function. These results demonstrate a bidirectional cross-talk between OPC and the surrounding neuronal network [24, 25]. The intracellular domain can be cleaved by the gamma-secretase and may influence the expression of genes, such as prostaglandin D2 synthase which has neuromodulatory properties [26]. NG2 has been reported to promote migration and proliferation in oligodendroglial and neoplastic glioma cells [27–30]. In OPC the effect on migration is mediated via modulation of Rho GTPases and RAC activity and an influence on cell polarity via selective subcellular localization [31, 32]. Contradictory results have been published with respect to the effect of NG2 on de- and remyelination, as well as on inflammation in inflammatory and/or demyelinating animal models. Mice lacking NG2 were reported to show reduced inflammation as well as reduced myelin damage and repair after injection of lysolecithin [33]. In contrast, in EAE experiments using this same NG2-/- mouse line [34] no differences in disease course or extent of de- and remyelination or inflammation was observed [35]. We hypothesized that the effect of a lack of NG2 might be amplified by extended time periods of demyelination. Furthermore, the initial NG2-/- mouse line [34] was generated by insertion of a neo cassette that may affect the function of nearby genes. We thus utilized mice lacking NG2 in which eYFP was inserted in the endogenous NG2 locus [36]. When bred to homozygosity these mice lack expression of NG2. We fed homozygous NG2-/- mice and their wild type littermates (NG2+/+) for 10 weeks with the copper chelator cuprizone which leads to oligodendroglial death and compared the extent of de- and remyelination as well as the degree of inflammation as measured by the numbers of Mac3 (+) microglia/macrophages. In addition, we isolated OPC from NG2-/- as well NG2+/+ mice to compare and analyze oligodendroglial properties prerequisite for remyelination, namely proliferation, migration and differentiation. In vitro, NG2-/- OPC demonstrated an increased migratory capacity in PDGF-AA, but not FGF-elicited chemotaxis; however lack of NG2 did not affect proliferation or differentiation of isolated OPC. No effect of NG2 deficiency on de- or remyelination, numbers of myelinated axons, oligodendrocytes or microglia/macrophages was observed.

BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions

BCAS1 expression identifies newly formed and actively myelinating oligodendrocytes in development, adulthood, and disease. Mapping active myelination with BCAS1 Neuronal axon demyelination causes motor and cognitive impairments in multiple sclerosis (MS) and other demyelinating disorders. Although remyelinating strategies have been proposed, the lack of markers to detect areas of active myelination hampers the development of effective therapies. Fard et al. show that myelinating oligodendrocytes constitute a unique population expressing breast carcinoma amplified sequence 1 (BCAS1) in rodent and human brain tissue. In brain samples from deceased MS patients, BCAS1+ cells are present around lesions, suggesting that remyelination might occur during MS and that BCAS1 expression could be used to track responses to remyelinating compounds for treating demyelinating disorders. Investigations into brain function and disease depend on the precise classification of neural cell types. Cells of the oligodendrocyte lineage differ greatly in their morphology, but accurate identification has thus far only been possible for oligodendrocyte progenitor cells and mature oligodendrocytes in humans. We find that breast carcinoma amplified sequence 1 (BCAS1) expression identifies an oligodendroglial subpopulation in the mouse and human brain. These cells are newly formed, myelinating oligodendrocytes that segregate from oligodendrocyte progenitor cells and mature oligodendrocytes and mark regions of active myelin formation in development and in the adult. We find that BCAS1+ oligodendrocytes are restricted to the fetal and early postnatal human white matter but remain in the cortical gray matter until old age. BCAS1+ oligodendrocytes are reformed after experimental demyelination and found in a proportion of chronic white matter lesions of patients with multiple sclerosis (MS) even in a subset of patients with advanced disease. Our work identifies a means to map ongoing myelin formation in health and disease and presents a potential cellular target for remyelination therapies in MS.

Publisher Correction: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

The originally published version of this Article omitted Tanja Kuhlmann and Michael Wegner as jointly supervising authors. This has now been corrected in both the PDF and HTML versions of the Article.