Combining physical & cognitive training with iPSC-derived dopaminergic neuron transplantation promotes graft integration & better functional outcome in parkinsonian marmosets.

Parkinson's disease (PD) is a relentlessly progressive and currently incurable neurodegenerative disease with significant unmet medical needs. Since PD stems from the degeneration of midbrain dopaminergic (DA) neurons in a defined brain location, PD patients are considered optimal candidates for cell replacement therapy. Clinical trials for cell transplantation in PD are beginning to re-emerge worldwide with a new focus on induced pluripotent stem cells (iPSCs) as a source of DA neurons since they can be derived from adult somatic cells and produced in large quantities under current good manufacturing practices. However, for this therapeutic strategy to be realized as a viable clinical option, fundamental translational challenges need to be addressed including the manufacturing process, purity and efficacy of the cells, the method of delivery, the extent of host reinnervation and the impact of patient-centered adjunctive interventions. In this study we report on the impact of physical and cognitive training (PCT) on functional recovery in the nonhuman primate (NHP) model of PD after cell transplantation. We observed that at 6 months post-transplant, the PCT group returned to normal baseline in their daily activity measured by actigraphy, significantly improved in their sensorimotor and cognitive tasks, and showed enhanced synapse formation between grafted cells and host cells. We also describe a robust, simple, efficient, scalable, and cost-effective manufacturing process of engraftable DA neurons derived from iPSCs. This study suggests that integrating PCT with cell transplantation therapy could promote optimal graft functional integration and better outcome for patients with PD.

LRRK2 Attenuates Antioxidant Response in Familial Parkinson's Disease Derived Neural Stem Cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.

Human Induced Pluripotent Stem Cell Phenotyping and Preclinical Modeling of Familial Parkinson's Disease.

Parkinson's disease (PD) is primarily idiopathic and a highly heterogenous neurodegenerative disease with patients experiencing a wide array of motor and non-motor symptoms. A major challenge for understanding susceptibility to PD is to determine the genetic and environmental factors that influence the mechanisms underlying the variations in disease-associated traits. The pathological hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra pars compacta region of the brain and post-mortem Lewy pathology, which leads to the loss of projecting axons innervating the striatum and to impaired motor and cognitive functions. While the cause of PD is still largely unknown, genome-wide association studies provide evidence that numerous polymorphic variants in various genes contribute to sporadic PD, and 10 to 15% of all cases are linked to some form of hereditary mutations, either autosomal dominant or recessive. Among the most common mutations observed in PD patients are in the genes LRRK2, SNCA, GBA1, PINK1, PRKN, and PARK7/DJ-1. In this review, we cover these PD-related mutations, the use of induced pluripotent stem cells as a disease in a dish model, and genetic animal models to better understand the diversity in the pathogenesis and long-term outcomes seen in PD patients.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

Creating Cell Model 2.0 Using Patient Samples Carrying a Pathogenic Mitochondrial DNA Mutation: iPSC Approach for LHON.

Leber's Hereditary Optic Neuropathy is the most prevalent mitochondrial neurological disease caused by mutations in mitochondrial DNA encoded respiratory complex I subunits. Although the genetic origin for Leber's hereditary optic neuropathy was identified about 30 years ago, the underlying pathogenesis is still unclear primarily due to the lack of a relevant system or cell model. Current models are limited to lymphoblasts, fibroblasts, or cybrid cell lines. As the disease phenotype is limited to retinal ganglion cells, induced pluripotent stem cells will serve as an excellent model for studying this tissue-specific disease, elucidating its underlying molecular mechanisms, and identifying novel therapeutic targets. Here, we describe a detailed protocol for the generation of retinal ganglion cells, and also cardiomyocytes for proof of iPSC pluripotency.

Dopamine D3 receptor ligand suppresses the expression of levodopa-induced dyskinesia in nonhuman primate model of parkinson's disease.

Parkinson's disease (PD) is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. The incidence of PD increases with aging and the expected burden will continue to escalate with our aging population. Since its discovery in the 1961 levodopa has remained the gold standard pharmacotherapy for PD. However, the progressive nature of the neurodegenerative process in and beyond the nigrostriatal system causes a multitude of side effects, including levodopa-induced dyskinesia within 5 years of therapy. Attenuating dyskinesia has been a significant challenge in the clinical management of PD. We report on a small molecule that eliminates the expression of levodopa-induced dyskinesia and significantly improves PD-like symptoms. The lead compound PD13R we discovered is a dopamine D3 receptor partial agonist with high affinity and selectivity, orally active and with desirable drug-like properties. Future studies are aimed at developing this lead compound for treating PD patients with dyskinesia.

Isolation and Purification of Self-Renewable Human Neural Stem Cells from iPSCs for Cell Therapy in Experimental Model of Ischemic Stroke.

Neural stem cell therapy has been galvanized by the discovery of pluripotent stem cells. The possibility to generate specialized central nervous system-specific differentiated cells using human somatic cells engineered to become induced pluripotent stem cells (iPSCs) was a game changer. This technology has broad applications in the field of regenerative medicine, in vitro disease modeling, targeted drug discovery, and precision medicine. Currently, iPSCs are one of the most promising cell sources amenable for commercialization and off-the-shelf neural stem cell therapy products. iPSCs exhibit a strong self-renewable ability that supports the development of a virtually unlimited source of neural cells for structural repair in neurological disorders. However, along with this strong proliferative capacity of iPSCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous population of human neural stem cells (hNSCs) are of paramount importance to ensure consistency in the composition of the cellular product and to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from iPSCs through the use of specific growth medium and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we will describe methods to analyze the ischemic stroke and size of grafts using magnetic resonance imaging and OsiriX software and neuroanatomical tracing procedures to study axonal remodeling after ischemic stroke and cell transplantation.

Coat Color-Facilitated Efficient Generation and Analysis of a Mouse Model of Down Syndrome Triplicated for All Human Chromosome 21 Orthologous Regions.

Down syndrome (DS) is one of the most complex genetic disorders in humans and a leading genetic cause of developmental delays and intellectual disabilities. The mouse remains an essential model organism in DS research because human chromosome 21 (Hsa21) is orthologously conserved with three regions in the mouse genome. Recent studies have revealed complex interactions among different triplicated genomic regions and Hsa21 gene orthologs that underlie major DS phenotypes. Because we do not know conclusively which triplicated genes are indispensable in such interactions for a specific phenotype, it is desirable that all evolutionarily conserved Hsa21 gene orthologs are triplicated in a complete model. For this reason, the Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+ mouse is the most complete model of DS to reflect gene dosage effects because it is the only mutant triplicated for all Hsa21 orthologous regions. Recently, several groups have expressed concerns that efforts needed to generate the triple compound model would be so overwhelming that it may be impractical to take advantage of its unique strength. To alleviate these concerns, we developed a strategy to drastically improve the efficiency of generating the triple compound model with the aid of a targeted coat color, and the results confirmed that the mutant mice generated via this approach exhibited cognitive deficits.

Age-related cognitive decline in baboons: modeling the prodromal phase of Alzheimer's disease and related dementias.

The aging of brain cells and synaptic loss are the major underlying pathophysiological processes contributing to the progressive decline in cognitive functions and Alzheimer's disease. The difference in cognitive performances observed between adult and aged subjects across species highlights the decline of brain systems with age. The inflection point in age-related cognitive decline is important for our understanding of the pathophysiology of neurodegenerative diseases and for timing therapeutic interventions. Humans and nonhuman primates share many similarities including age-dependent changes in gene expression and decline in neural and immune functions. Given these evolutionary conserved organ systems, complex human-like behavioral and age-dependent changes may be modeled and monitored longitudinally in nonhuman primates. We integrated three clinically relevant outcome measures to investigate the effect of age on cognition, motor function and diurnal activity in aged baboons. We provide evidence of a naturally-occurring age-dependent precipitous decline in movement planning, in learning novel tasks, in simple discrimination and in motivation. These results suggest that baboons aged ~20 years (equivalent to ~60 year old humans) may offer a relevant model for the prodromal phase of Alzheimer's disease and related dementias to investigate mechanisms involved in the precipitous decline in cognitive functions and to develop early therapeutic interventions.

Non-cell autonomous mechanism of Parkinson's disease pathology caused by G2019S LRRK2 mutation in Ashkenazi Jewish patient: Single cell analysis.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of the midbrain dopaminergic neurons, which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease, however about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2) with high prevalence in Ashkenazi Jewish patients and in North African Berber and Arab patients. It is still not fully understood how this mutation leads to PD pathology. In this study, we derived induced pluripotent stem cells (iPSCs) from an Ashkenazi Jewish patient with G2019S LRRK2 mutation to isolate self-renewable multipotent neural stem cells (NSCs) and to model this form of PD in vitro. To investigate the cellular diversity and disease pathology in the NSCs, we used single cell RNA-seq transcriptomic profiling. The evidence suggests there are three subpopulations within the NSCs: a committed neuronal population, intermediate stage population and undifferentiated stage population. Unbiased single-cell transcriptomic analysis revealed differential expression and dysregulation of genes involved in PD pathology. The significantly affected genes were involved in mitochondrial function, DNA repair, protein degradation, oxidative stress, lysosome biogenesis, ubiquitination, endosome function, autophagy and mitochondrial quality control. The results suggest that G2019S LRRK2 mutation may affect multiple cell types in a non-cell autonomous mechanism of PD pathology and that unbiased single-cell transcriptomics holds promise for personalized medicine.

Generation of Neural Stem Cells from Induced Pluripotent Stem Cells.

Neural stem cells (NSCs) are defined by three necessary but not sufficient criteria: (1) self-renewable, (2) ability to generate a large number of progeny, and (3) ability to differentiate into the principal central nervous system (CNS) cell types, neurons, astrocytes, and oligodendrocytes. There are various approaches to derive neural lineages from pluripotent stem cells. It is well recognized that the chosen method of NSC derivation is critical to answering the basic biology question under investigation, to the success rate in drug discovery and to the efficacy of the therapeutic cells intended for repairing the CNS. There are three critical attributes of NSCs: (1) well-defined and stable cellular composition, (2) consistent process of perpetuation that avoids drift in composition, and (3) stable phenotype or therapeutic activity of the NSCs or their differentiated progeny. Over the past decades, we have been continuously developing consistent processes for generating stable, multipotent self-renewable NSCs from various sources. In this chapter, we report a method to generate NSCs from induced pluripotent stem cells.

Differentiation of Neural Stem Cells Derived from Induced Pluripotent Stem Cells into Dopaminergic Neurons.

Dopaminergic (DA) neurons are involved in many critical functions within the central nervous system (CNS), and dopamine neurotransmission impairment underlies a wide range of disorders from motor control deficiencies, such as Parkinson's disease (PD), to psychiatric disorders, such as alcoholism, drug addictions, bipolar disorders, schizophrenia and depression. Neural stem cell-based technology has potential to play an important role in developing efficacious biological and small molecule therapeutic products for disorders with dopamine dysregulation. Various methods of differentiating DA neurons from pluripotent stem cells have been reported. In this chapter, we describe a simple technique using dopamine-inducing factors (DIFs) to differentiate neural stem cells (NSCs), isolated from induced pluripotent stem cells (iPSCs) into DA neurons.

Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.

Genetic reporters offer attractive approaches to investigate gene expression, gene function, and spatiotemporal assessment in vitro and in vivo. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of the dopamine neurotransmitter, and thus it has been used as a reliable marker for dopaminergic neurons in vitro and in vivo. Herein we describe a method for making iPSC lines with TH-green fluorescent protein reporter gene using CRISPR/Cas9 technique.

Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells.

Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP beads.

Bioinformatics Analysis of Single-Cell RNA-Seq Raw Data from iPSC-Derived Neural Stem Cells.

This chapter describes a pipeline for basic bioinformatics analysis of single-cell sequencing data (see Chap. 10 : Single-Cell Library Preparation). Starting with raw sequencing data, we describe how to quality check samples, to create an index from a reference genome, to align the sequences to an index, and to quantify transcript abundances. The curated data sets will enable differential expression analysis, population analysis, and pathway analysis.

Assay for Assessing Mitochondrial Function in iPSC-Derived Neural Stem Cells and Dopaminergic Neurons.

Rapid and reliable assessment of mitochondrial bioenergetics is a vital tool in drug discovery studies aimed at reversing or improving mitochondrial dysfunction. Induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) carry and replicate the donor disease pathology and can be an ideal cellular model for phenotypic screening of compounds. Herein we describe the use of Seahorse XFe96 analyzer to assess mitochondrial functions in iPSC-derived NSCs for drug screening.

Reference Transcriptome for Deriving Marmoset Induced Pluripotent Stem Cells.

Limited access to primary tissue from various nonhuman primate (NHP) species represents a significant unmet need that hampers progress in understanding unique cellular diversity and gene regulation of specific tissues and organs in stem cell translational research. Most comparative biology studies have been limited to using postmortem tissue usually frozen specimens with limited utility for research. The generation of induced pluripotent stem cell (iPSC) lines from somatic cells, such as adult skin or blood cells, offers an alternative to invasive and ethically controversial interventions for acquiring tissue. Pluripotent iPSCs have virtually an unlimited capacity to proliferate and differentiate into all cell types of the body. We are generating high-quality validated NHP iPSC lines to offer to scientific community and facilitate their research programs. We use the non-integrative episomal vector system to generate iPSCs from NHP skin biopsies. In this chapter we describe the validation of NHP iPSC lines by confirming pluripotency and their propensity to differentiate into all three germ layers ectoderm, mesoderm, and endoderm according to established standards and measurable limits for a set of marker genes incorporated into a scorecard.

Isolation and Differentiation of Self-Renewable Neural Stem Cells from Marmoset-Induced Pluripotent Stem Cells.

Neural stem cells (NSCs) are multipotent and self-renewing precursor cells that give rise to all cell types of the central nervous system (CNS). They can be used for modeling CNS in vitro, for developmental studies and for cell replacement therapies. NSCs can be derived from pluripotent stem cells through differentiation using specific growth factors. Nonhuman primates (NHP) are critical preclinical models for translational research. Induced pluripotent stem cells (iPSCs) can be generated from NHP for the purposes of allogenic or autologous cell replacement studies. Here, we describe the derivation of NSCs from NHP iPSCs.

Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage.

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

Charting the onset of Parkinson-like motor and non-motor symptoms in nonhuman primate model of Parkinson's disease.

Parkinson's disease is a progressive neurodegenerative disease increasingly affecting our aging population. Remarkable advances have been made in developing novel therapies to control symptoms, halt or cure the disease, ranging from physiotherapy and small molecules to cell and gene therapy. This progress was enabled by the existence of reliable animal models. The nonhuman primate model of Parkinson's disease emulates the cardinal symptoms of the disease, including tremor, rigidity, bradykinesia, postural instability, freezing and cognitive impairment. However, this model is established through the specific loss of midbrain dopaminergic neurons, while our current knowledge reflects the reality of Parkinson's disease as a multisystem disease. Parkinson's disease involves both motor and non-motor symptoms, such as sleep disturbance, olfaction, gastrointestinal dysfunctions, depression and cognitive deficits. Some of the non-motor symptoms emerge earlier at the prodromal phase and worsen with disease progression, yet in basic and translational studies, they are rarely considered as endpoints. In this study, we set to characterize an ensemble of less described motor and non-motor dysfunctions in the marmoset MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model. We provide evidence that this animal model expresses postural head tremor and a progressive worsening of fine motor skills, movement coordination and cognitive abilities over a 6-month period. We report for the first time a non-invasive approach showing detailed analysis of daytime and nighttime sleep and circadian rhythm disturbance remarkably similar to Parkinson's disease patients. This study describes the incidence of tremors, motor and non-motor dysfunctions in a preclinical model and highlights the need for their consideration in translating effective new therapeutic approaches for Parkinson's disease.