Showing papers by "Clive N. Svendsen published in 2021"

Cross-Comparison of Human iPSC Motor Neuron Models of Familial and Sporadic ALS Reveals Early and Convergent Transcriptomic Disease Signatures.

Abstract: Induced pluripotent stem cell (iPSC)-derived neural cultures from amyotrophic lateral sclerosis (ALS) patients can model disease phenotypes. However, heterogeneity arising from genetic and experimental variability limits their utility, impacting reproducibility and the ability to track cellular origins of pathogenesis. Here, we present methodologies using single-cell RNA sequencing (scRNA-seq) analysis to address these limitations. By repeatedly differentiating and applying scRNA-seq to motor neurons (MNs) from healthy, familial ALS, sporadic ALS, and genome-edited iPSC lines across multiple patients, batches, and platforms, we account for genetic and experimental variability toward identifying unified and reproducible ALS signatures. Combining HOX and developmental gene expression with global clustering, we anatomically classified cells into rostrocaudal, progenitor, and postmitotic identities. By relaxing statistical thresholds, we discovered genes in iPSC-MNs that were concordantly dysregulated in postmortem MNs and yielded predictive ALS markers in other human and mouse models. Our approach thus revealed early, convergent, and MN-resolved signatures of ALS.

Emerging technologies and their impact on regulatory science.

Abstract: There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care...

Oligodendrocyte progenitor cell maturation is dependent on dual function of MCT8 in the transport of thyroid hormone across brain barriers and the plasma membrane.

Abstract: Inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) causes a rare and debilitating form of X-linked psychomotor disability known as Allan Herndon Dudley syndrome (AHDS). One of the most prominent pathophysiological symptoms of MCT8-deficiency is hypomyelination. Here, patient-derived induced pluripotent stem cells (iPSCs) were used to study the role of MCT8 and TH on the maturation of oligodendrocytes. Interestingly, neither MCT8 mutations nor reduced TH affected the in vitro differentiation of control or MCT8-deficient iPSCs into oligodendrocytes. To assess whether patient-derived iPSC-derived oligodendrocyte progenitor cells (iOPCs) could provide myelinating oligodendrocytes in vivo, cells were transplanted into the shiverer mouse corpus callosum where they survived, migrated, and matured into myelinating oligodendrocytes, though the myelination efficiency was reduced compared with control cells. When MCT8-deficient and healthy control iOPCs were transplanted into a novel hypothyroid immunodeficient triple knockout mouse (tKO, mct8-/- ; oatp1c1-/- ; rag2-/- ), they failed to provide behavioral recovery and did not mature into oligodendrocytes in the hypothyroid corpus callosum, demonstrating the critical role of TH transport across brain barriers in oligodendrocyte maturation. We conclude that MCT8 plays a cell autonomous role in oligodendrocyte maturation and that functional TH transport into the central nervous system will be required for developing an effective treatment for MCT8-deficient patients.

The proSAAS chaperone provides neuroprotection and attenuates transsynaptic α-synuclein spread in rodent models of Parkinson's disease

Abstract: Parkinson’s disease is a devastating motor disorder involving the aberrant aggregation of the synaptic protein synuclein (aSyn) and degeneration of the nigrostriatal dopaminergic tract. We previously showed that proSAAS, a small secreted chaperone protein widely expressed in neurons within the brain, is able to block aSyn-induced dopaminergic cytotoxicity in primary nigral neuron cultures. We show here that coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This positive functional outcome was accompanied by significant amelioration of the human aSyn-induced loss of both nigral tyrosine hydroxylase-positive cells and striatal tyrosine hydroxylase-positive terminals, demonstrating clear proSAAS-mediated protection of the nigro-striatal tract. ProSAAS overexpression also reduced the content of human aSyn protein in both the nigra and striatum and reduced the loss of tyrosine hydroxylase protein in both regions. Since proSAAS is a secreted protein, we tested the possibility that proSAAS is able to block the transsynaptic spread of aSyn from the periphery to the central nervous system, increasingly recognized as a potentially significant pathological mechanism. The number of human aSyn-positive neurites in the pons and caudal midbrain of mice following administration of human aSyn-encoding AAV into the vagus nerve was considerably reduced in mice coinjected with proSAAS-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. We suggest that proSAAS may represent a promising target for therapeutic development in Parkinson’s disease. Significance This paper describes two independent avenues of research that both provide support for the in vivo neuroprotective function of this small chaperone protein. In the first approach, we show that proSAAS overexpression provides remarkably effective protection against dopaminergic neurotoxicity in a rat model of Parkinson’s disease. This conclusion is supported both by three independent assays of motor function as well as by quantitative analysis of surviving dopaminergic neurons in brain areas involved in the control of motor function. In the second line of research, we show that in mice, the spread of human synuclein across synapses can be blunted by proSAAS overexpression.

Generation of iPSC lines with high cytogenetic stability from peripheral blood mononuclear cells (PBMCs)

Abstract: The utility of human induced pluripotent stem cells (hiPSCs) is contingent upon genomic integrity and stability. Recurrent genomic aberrations have been observed in human iPSC lines upon long-term culture, [~]10-25% demonstrate karyotype abnormalities. We describe a new and reliable non-integrating episomal plasmid reprogramming method for fresh (unexpanded) peripheral blood mononuclear cells (PBMC) into iPSCs (PBMC-iPSCs). PBMC-iPSCs produced using this method have a superior chromosome-level karyotype stability rate ([~]5% abnormality rate for all chromosomes; 2.8% for autosomes). After extended culture PBMC-iPSCs maintain a low rate of abnormalities (2% for autosomes). Deep coverage whole genome sequencing in a subset of PBMC-iPSC lines showed no shared single nucleotide polymorphisms (SNPs) or structural variants are introduced during reprogramming and maintenance of PBMC-iPSCs. iPSCs reprogrammed from unexpanded PBMCs have consistently high cytogenetic stability and minimal genomic aberrations, suggesting this method is highly suited for iPSCs in research and therapeutic clinical applications.

A novel treatment for Parkinson's disease and ALS: Combined cell and gene therapies

Abstract: Stem cell and gene therapy approaches for diseases of the brain are rapidly evolving. This chapter describes how one approach moved from basic lab studies into clinical trials for Parkinson's and ALS. Pioneering work using human fetal tissue in Parkinson's disease laid the foundations for stem cell approaches where there was better control of the cellular product. Astrocytes proved more realistic in transplantation studies and the ability to engineer them to release powerful growth factors such as ALS using gene therapy vectors increased their efficacy. The final closing sections move the reader through the FDA process and describe a full clinical trial that has recently completed.

AAV9-MCT8 delivery at juvenile stage ameliorates neurological and behavioral deficits in an Allan-Herndon-Dudley Syndrome mouse model

Abstract: Allan-Herndon-Dudley syndrome (AHDS) is a severe X-linked intellectual and psychomotor disability disorder accompanied by abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in the monocarboxylate transporter 8 (MCT8), a specific TH transporter widely expressed in the central nervous system. MCT8 gene mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. AAV9-based gene therapy is a promising approach to treat monogenic neurological disorders. Here, the potential of this approach was tested in the well-established double knockout (dKO) Mct8-/y; Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/- mouse model of AHDS, which displays disease-relevant neurological and TH phenotypes. Systemic intravenous delivery of adeno-associated virus serotype 9 (AAV9)-MCT8 at a juvenile stage led to improved locomotor and cognitive function, as well as rescue of T3-brain content and T3-related gene expression. This preclinical study indicates that this gene therapy may improve the neurological symptoms of AHDS patients.

Human pluripotent stem cell derived neurodegenerative disease models on a microfluidic chip

Abstract: Described herein is a microphysiological system for models of disease. Specifically, induced pluripotent stem cells (iPSCs) and iPSC-derived cells, including those obtained from disease patients, are seeded onto microfluidic “chip” devices to study cellular development and disease pathogenesis. Herein, neurodegenerative disease modeling, including Parkinson's Disease (PD) is shown to reproduce key PD pathology in a vascularized human model that contains neurons relating to PD pathology. Such compositions and methods are used for research for PD biomarkers, patient screening for PD risk assessment, and therapeutic discovery and testing. A panel of biomarkers are generated through analysis of living PD-chips by neural activity, whole transcriptomic, proteomic, and metabolomic analysis, and functional enzyme tests of media and tissue. Introducing therapeutics through a vasculature channel, coupled with blood brain barrier penetration studies can be assessed for efficacy in the human neural cells present in the PD-Chip.

Diagnostic and drug screening for molecular signatures of early onset sporadic parkinson's disease

Abstract: Induced Pluripotent Stem Cell (Ipsc) technology enables the generation and study of living brain tissue relevant to Parkinson's disease (PD) ex vivo. Utilizing cell lines from PD patients presents a powerful discovery system that links cellular phenotypes observed in vitro with real clinical data. Differentiating patient-derived iPSCs towards a dopaminergic (DA) neural fate revealed that these cells exhibit molecular and functional properties of DA neurons in vitro that are observed to significantly degenerate in the substantia nigra of PD patients. Clinical symptoms that drive the generation of other relevant cell types may also yield novel PD-specific phenotypes in vitro that have the potential to lead to new therapeutic avenues for patients with PD. Due to their early onset and non-familial origin, differentiated nervous tissue from these patients offer a key opportunity to discover neuron subtype-specific pathological mechanisms and importantly interrogate the contribution of their genetic background in susceptibility to PD.

Pkc pathway in parkinson's disease

Abstract: Induced Pluripotent Stem Cell (Ipsc) technology enables the generation and study of living brain tissue relevant to Parkinson's disease (PD) ex vivo. Utilizing cell lines from PD patients presents a powerful discovery system that links cellular phenotypes observed in vitro with real clinical data. Differentiating patient-derived iPSCs towards a dopaminergic (DA) neural fate revealed that these cells exhibit molecular and functional properties of DA neurons in vitro that are observed to significantly degenerate in the substantia nigra of PD patients. Clinical symptoms that drive the generation of other relevant cell types may also yield novel PD-specific phenotypes in vitro that have the potential to lead to new therapeutic avenues for patients with PD. Due to their early onset and non-familial origin, differentiated nervous tissue from these patients offer a key opportunity to discover neuron subtype-specific pathological mechanisms and importantly interrogate the contribution of their genetic background in susceptibility to PD.