Showing papers by "Clive N. Svendsen published in 2017"

The Library of Integrated Network-Based Cellular Signatures NIH Program: System-Level Cataloging of Human Cells Response to Perturbations

Abstract: The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

Abstract: The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843.

Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice

Abstract: Neural cultures derived from Huntington’s disease (HD) patient-derived induced pluripotent stem cells were used for ‘omics’ analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time.

Enhanced Utilization of Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids Using Microengineered Chips.

Abstract: Background and Aims Human intestinal organoids derived from induced pluripotent stem cells have tremendous potential to elucidate the intestinal epithelium’s role in health and disease, but it is difficult to directly assay these complex structures. This study sought to make this technology more amenable for study by obtaining epithelial cells from induced pluripotent stem cell–derived human intestinal organoids and incorporating them into small microengineered Chips. We then investigated if these cells within the Chip were polarized, had the 4 major intestinal epithelial subtypes, and were biologically responsive to exogenous stimuli. Methods Epithelial cells were positively selected from human intestinal organoids and were incorporated into the Chip. The effect of continuous media flow was examined. Immunocytochemistry and in situ hybridization were used to demonstrate that the epithelial cells were polarized and possessed the major intestinal epithelial subtypes. To assess if the incorporated cells were biologically responsive, Western blot analysis and quantitative polymerase chain reaction were used to assess the effects of interferon (IFN)-γ, and fluorescein isothiocyanate–dextran 4 kDa permeation was used to assess the effects of IFN-γ and tumor necrosis factor-α on barrier function. Results The optimal cell seeding density and flow rate were established. The continuous administration of flow resulted in the formation of polarized intestinal folds that contained Paneth cells, goblet cells, enterocytes, and enteroendocrine cells along with transit-amplifying and LGR5+ stem cells. Administration of IFN-γ for 1 hour resulted in the phosphorylation of STAT1, whereas exposure for 3 days resulted in a significant upregulation of IFN-γ related genes. Administration of IFN-γ and tumor necrosis factor-α for 3 days resulted in an increase in intestinal permeability. Conclusions We demonstrate that the Intestine-Chip is polarized, contains all the intestinal epithelial subtypes, and is biologically responsive to exogenous stimuli. This represents a more amenable platform to use organoid technology and will be highly applicable to personalized medicine and a wide range of gastrointestinal conditions.

Concise Review: Stem Cells for Corneal Wound Healing

Abstract: Corneal wound healing is a complex process that occurs in response to various injuries and commonly used refractive surgery. It is a significant clinical problem, which may lead to serious complications due to either incomplete (epithelial) or excessive (stromal) healing. Epithelial stem cells clearly play a role in this process, whereas the contribution of stromal and endothelial progenitors is less well studied. The available evidence on stem cell participation in corneal wound healing is reviewed, together with the data on the use of corneal and non-corneal stem cells to facilitate this process in diseased or postsurgical conditions. Important aspects of corneal stem cell generation from alternative cell sources, including pluripotent stem cells, for possible transplantation upon corneal injuries or in disease conditions are also presented. Stem Cells 2017;35:2105-2114.

A Comparison of mRNA Sequencing with Random Primed and 3'-Directed Libraries.

Abstract: Creating a cDNA library for deep mRNA sequencing (mRNAseq) is generally done by random priming, creating multiple sequencing fragments along each transcript. A 3'-end-focused library approach cannot detect differential splicing, but has potentially higher throughput at a lower cost, along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) that correct PCR bias. Here, we compare an implementation of such a 3'-digital gene expression (3'-DGE) approach with "conventional" random primed mRNAseq. Given our particular datasets on cultured human cardiomyocyte cell lines, we find that, while conventional mRNAseq detects ~15% more genes and needs ~500,000 fewer reads per sample for equivalent statistical power, the resulting differentially expressed genes, biological conclusions, and gene signatures are highly concordant between two techniques. We also find good quantitative agreement at the level of individual genes between two techniques for both read counts and fold changes between given conditions. We conclude that, for high-throughput applications, the potential cost savings associated with 3'-DGE approach are likely a reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable many larger scale studies focusing on not only differential expression analysis, but also quantitative transcriptome profiling.

Directed Differentiation of Human Induced Pluripotent Stem Cells into Fallopian Tube Epithelium.

Abstract: The fallopian tube epithelium (FTE) has been recognized as a site of origin of high-grade serous ovarian cancer (HGSC). However, the absence of relevant in vitro human models that can recapitulate tissue-specific architecture has hindered our understanding of FTE transformation and initiation of HGSC. Here, induced pluripotent stem cells (iPSCs) were used to establish a novel 3-dimensional (3D) human FTE organoid in vitro model containing the relevant cell types of the human fallopian tube as well as a luminal architecture that closely reflects the organization of fallopian tissues in vivo. Modulation of Wnt and BMP signaling directed iPSC differentiation into Mullerian cells and subsequent use of pro-Mullerian growth factors promoted FTE precursors. The expression and localization of Mullerian markers verified correct cellular differentiation. An innovative 3D growth platform, which enabled the FTE organoid to self-organize into a convoluted luminal structure, permitted matured differentiation to a FTE lineage. This powerful human-derived FTE organoid model can be used to study the earliest stages of HGSC development and to identify novel and specific biomarkers of early fallopian tube epithelial cell transformation.

Ex vivo gene therapy for the treatment of neurological disorders.

Abstract: Ex vivo gene therapy involves the genetic modification of cells outside of the body to produce therapeutic factors and their subsequent transplantation back into patients. Various cell types can be genetically engineered. However, with the explosion in stem cell technologies, neural stem/progenitor cells and mesenchymal stem cells are most often used. The synergy between the effect of the new cell and the additional engineered properties can often provide significant benefits to neurodegenerative changes in the brain. In this review, we cover both preclinical animal studies and clinical human trials that have used ex vivo gene therapy to treat neurological disorders with a focus on Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, and stroke. We highlight some of the major advances in this field including new autologous sources of pluripotent stem cells, safer ways to introduce therapeutic transgenes, and various methods of gene regulation. We also address some of the remaining hurdles including tunable gene regulation, in vivo cell tracking, and rigorous experimental design. Overall, given the current outcomes from researchers and clinical trials, along with exciting new developments in ex vivo gene and cell therapy, we anticipate that successful treatments for neurological diseases will arise in the near future.

Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1G93A ALS rat but has adverse side effects.

Abstract: Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1 G93A ALS rat but has adverse side effects

Ferumoxytol Labeling of Human Neural Progenitor Cells for Diagnostic Cellular Tracking in the Porcine Spinal Cord With Magnetic Resonance Imaging

Abstract: We report on the diagnostic capability of magnetic resonance imaging (MRI)-based tracking of ferumoxytol-labeled human neural progenitor cells (hNPCs) transplanted into the porcine spinal cord. hNPCs prelabeled with two doses of ferumoxytol nanoparticles (hNPC-FLow and hNPC-FHigh ) were injected into the ventral horn of the spinal cord in healthy minipigs. Ferumoxytol-labeled grafts were tracked in vivo up to 105 days after transplantation with MRI. Injection accuracy was assessed in vivo at day 14 and was predictive of "on" or "off" target cell graft location assessed by histology. No difference in long-term cell survival, assessed by quantitative stereology, was observed among hNPC-FLow , hNPC-FHigh , or control grafts. Histological iron colocalized with MRI signal and engrafted human nuclei. Furthermore, the ferumoxytol-labeled cells retained nanoparticles and function in vivo. This approach represents an important leap forward toward facilitating translation of cell-tracking technologies to clinical trials by providing a method of assessing transplantation accuracy, delivered dose, and potentially cell survival. Stem Cells Translational Medicine 2017;6:139-150.

Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

Abstract: Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-syn in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. Recent studies show that murine neural stem cell (NSC) transplantation can improve cognitive or motor function in transgenic models of Alzheimer's and Huntington's disease, and DLB. However, examination of clinically relevant human NSCs in these models is hindered by the challenges of xenotransplantation and the confounding effects of immunosuppressant drugs on pathology and behavior. To address this challenge, we developed an immune-deficient transgenic model of DLB that lacks T-, B-, and NK-cells, yet exhibits progressive accumulation of human α-syn (h-α-syn)-laden inclusions and cognitive and motor impairments. We demonstrate that clinically relevant human neural progenitor cells (line CNS10-hNPCs) survive, migrate extensively and begin to differentiate preferentially into astrocytes following striatal transplantation into this DLB model. Critically, grafted CNS10-hNPCs rescue both cognitive and motor deficits after 1 and 3 months and, furthermore, restore striatal dopamine and glutamate systems. These behavioral and neurochemical benefits are likely achieved by reducing α-syn oligomers. Collectively, these results using a new model of DLB demonstrate that hNPC transplantation can impact a broad array of disease mechanisms and phenotypes and suggest a cellular therapeutic strategy that should be pursued. Stem Cells Translational Medicine 2017;6:1477-1490.

Systems and methods for growth of intestinal cells in microfluidic devices

Abstract: Organs-on-chips are microfluidic devices for culturing living cells in micrometer sized chambers in order to model physiological functions of tissues and organs. Engineered patterning and continuous fluid flow in these devices has allowed culturing of intestinal cells bearing physiologically relevant features and sustained exposure to bacteria while maintaining cellular viability, thereby allowing study of inflammatory bowl diseases. However, existing intestinal cells do not possess all physiologically relevant subtypes, do not possess the repertoire of genetic variations, or allow for study of other important cellular actors such as immune cells. Use of iPSC-derived epithelium, including IBD patient-specific cells, allows for superior disease modeling by capturing the multi-faceted nature of the disease.

Patients with ALS show highly correlated progression rates in left and right limb muscles.

Abstract: Objective: Amyotrophic lateral sclerosis (ALS) progresses at different rates between patients, making clinical trial design difficult and dependent on large cohorts of patients. Currently, there are few data showing whether the left and right limbs progress at the same or different rates. This study addresses rates of decline in specific muscle groups of patients with ALS and assesses whether there is a relationship between left and right muscles in the same patient, regardless of overall progression. Methods: A large cohort of patients was used to assess decline in muscle strength in right and left limbs over time using 2 different methods: The Tufts Quantitative Neuromuscular Exam and Accurate Test of Limb Isometric Strength protocol. Then advanced linear regression statistical methods were applied to assess progression rates in each limb. Results: This report shows that linearized progression models can predict general slopes of decline with good accuracy. Critically, the data demonstrate that while overall decline is variable, there is a high degree of correlation between left and right muscle decline in ALS. This implies that irrespective of which muscle starts declining soonest or latest, their rates of decline following onset are more consistent. Conclusions: First, this study demonstrates a high degree of power when using unilateral treatment approaches to detect a slowing in disease progression in smaller groups of patients, thus allowing for paired statistical tests. These findings will be useful in transplantation trials that use muscle decline to track disease progression in ALS. Second, these findings discuss methods, such as tactical selection of muscle groups, which can improve the power efficiency of all ALS clinical trials.

Development of spinal cord on a microfluidic chip

Abstract: The invention relates to culturing brain endothelial cells, and optionally astrocytes and neurons in a fluidic device under conditions whereby the cells mimic the structure and function of the blood brain barrier. Culture of such cells in a microfluidic device, whether alone or in combination with other cells, drives maturation and/or differentiation further than existing systems.

A Comparison of mRNA Sequencing with Random Primed and 3'-Directed Libraries

Abstract: Deep mRNA sequencing (mRNAseq) is the state-of-the-art for whole transcriptome measurements. A key step is creating a library of cDNA sequencing fragments from RNA. This is generally done by random priming, creating multiple sequencing fragments along the length of each transcript. A 39 end-focused library approach cannot detect differential splicing, but has potentially higher throughput at lower cost (~10-fold lower), along with the ability to improve quantification by using transcript molecule counting with unique molecular identifiers (UMI) to correct for PCR bias. Here, we compare implementation of such a 39-digital gene expression (39-DGE) approach with "conventional" random primed mRNAseq, which has not yet been done. We find that while conventional mRNAseq detects ~15% more genes, the resulting lists of differentially expressed genes and therefore biological conclusions and gene signatures are highly concordant between the two techniques. We also find good quantitative agreement on the level of individual genes between the two techniques in terms of both read counts and fold change between two conditions. We conclude that for high-throughput applications, the potential cost savings associated with the 39-DGE approach are a very reasonable tradeoff for modest reduction in sensitivity and inability to observe alternative splicing, and should enable much larger scale studies focused on not only differential expression analysis, but also quantitative transcriptome profiling. The computational scripts and programs, along with experimental standard operating procedures used in our pipeline presented here, are freely available on our website (www.dtoxs.org).

Huntington’s Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits

Abstract: Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.

Methods and compositions for spinal cord cells

Abstract: Described here are systems and methods for deriving both spinal motor neurons and brain microvascular endothelial cells from induced pluripotent stem cells using distinct methods and combining them in a chip format. Neurons cultured alone in chip microvolume displayed increased calcium transient function and chip-specific gene expression. When seeded with endothelial cells, interaction further enhanced neural function, elicited vascular-neural interaction, niche gene expression with enhanced in vivo-like signatures arising from the chip co-cultures. Development of novel media formulations further allow for improved readout of differentiation process, by eliminating additives that otherwise confound differentiation processes and resulting phenotypes.