2004 — 2008 |
Ming, Guo-Li |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Adaptation Mechanisms in Long-Range Growth Cone Guidance @ Johns Hopkins University
DESCRIPTION (provided by applicant): The intricate neural network formation during development is achieved by correct pathfinding of the tip of a growing axon, the growth cone, in response to guidance cues. Diffusible and substrate bound molecules presented in the environment can form gradients and function as long-range chemoattractants or chemorepellents to guide the growth cones to their targets. Over the last two decades, several families of guidance molecules and their receptors have been identified by genetic and biochemical approaches. The signal transduction and modulation mechanisms underlying the directed growth cone motility, however, have just begun to be elucidated. Our long-term goal is to understand the molecular and cellular mechanisms that determine the motility and directionality of developing and regenerating axons in response to guidance cues as well as inhibitory molecules and to develop therapeutic strategies to promote regeneration of specific axonal tracks after injury or diseases of human CNS. Growth cone guidance exemplifies the complexity of how a single cell interacts with its environment. The neuronal growth cones can respond with highly oriented polarity and motility toward the source of chemoattractant based on detecting a very shallow molecular gradient presented in the environment, which can be as low as 1%. However, a growth cone can also maintain its sensitivity when migrating up a gradient of guidance cue, the average concentration of which can span several orders of magnitude. A temporal mechanism must therefore be acquired by the growth cone that can reset the sensitivity of the growth cone during migration, which involves adaptation. We have developed a quantitative assay to analyze the steering decision of growth cones in a defined gradient of guidance cues. Using this approach with Xenopus spinal neurons in cultures, we have shown that the growth cones of these neurons exhibit consecutive phases of desensitization (loss of response) and resensitization (adaptation) when migrating up a gradient of netrin-1, an evolutionally conserved developmental guidance molecule essential for neural circuit formation in many organisms. In this proposal, we aim to investigate the molecular mechanisms underlying the adaptive growth cone responses to netrin-1. Specifically, we will focus on examining immediate signaling mechanisms associated with Deleted in Colorectal Cancer (DCC), a well characterized membrane receptors for netrin-1, with a combination of biochemistry, immunocytochemistry, Ca2+ imaging and growth cone turning assays
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1 |
2010 — 2014 |
Ming, Guo-Li |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Calcium Signaling in Neuronal Navigation @ Johns Hopkins University
DESCRIPTION (provided by applicant): Directed neuronal navigation, including both cell body migration and growth cone path-finding, is a pre- requisite for the establishment of the precisely wired neural network and is essential for the proper function of the brain. Accumulating evidence suggests that growth cone navigation and neuronal cell navigation during early development share many similar features, including responses to a similar set of guidance cues, activation of specific intracellular signaling cascades and cytoskeletal changes for directed movements. For example, netrin-1, an evolutionally conserved long-range growth cone guidance cue essential for neural circuit formation during development, also directs cell migration of cortical neurons and olfactory neurons. Ca2+ signaling has emerged as a central player in mediating growth cone and cellular responses to many guidance cues, including netrin-1. The spatial and temporal regulation of Ca2+ signaling underlying directed neuronal navigation, however, is not well understood. While neural network formation occurs predominantly during the prenatal and early postnatal periods, new neurons are continuously generated from neural progenitors and integrated into the existing neural network in discrete regions of adult mammalian brain, including the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. Neurodevelopment in the adult brain recapitulates the major neural developmental milestones, from proliferation and fate specification of neural progenitors, to neuronal morphogenesis, cell migration, axon and dendritic guidance, and synapse formation by neuronal progeny. Because adult neurogenesis occurs in a significantly different environment from embryonic neurogenesis, whether the molecular mechanisms underlying neural development are conserved is not clear. Our long-term goal is to understand the molecular and cellular mechanisms that determine the motility and directionality of developing neurons in response to guidance cues and to develop therapeutic strategies to promote regeneration after injury or diseases of the human central nervous system (CNS). In the current project, we aim to understand the role of Ca2+ signaling in regulating neuronal navigation during early neural development and in the adult brain with the central hypothesis that TRPC, STIM1 and Orai proteins co-operate to set the basal and induced Ca2+ levels for directed motility of growth cones and neurons, using a combination of in vitro growth cone turning assay, immunocytochemistry, multi-photon confocal microscopy and electrophysiology. Our study will provide important information on the molecular mechanisms underlying neuronal navigation and may lead to novel insights as to whether neuronal navigation processes are similarly or differentially regulated in the mature brain, which is important for developing strategies in promoting regeneration. PUBLIC HEALTH RELEVANCE: The project aims at understanding the functional roles of STIM1, TRPC and Orai proteins in regulating the calcium changes for directed growth cone guidance and neuronal cell migration during embryonic development and in the adult brain. Findings from these studies may lead to novel strategies to functionally replace damaged or lost neurons and to promote endogenous repair after injury or degenerative neurological disease.
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1 |
2010 — 2012 |
Ming, Guo-Li |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neurobiology of Mecp2 in Adult Neurogenesis @ Johns Hopkins University
Rett syndrome (RTT) is an X-linked dominant disorder caused by loss-of-function mutations in the gene encoding methyl CpG binding protein 2 (MECP2). Studies of human patient samples and animal models suggest that MECP2/MeCP2 may play essential roles In neuronal maturation and synapse formation/maintenance during development. The neurobiology of MeCP2 in neuronal development remains to be fully characterized, in the dentate gynjs of the hippocampus, new granule neurons are continuously generated from neural progenitors throughout life in all mammals examined, including humans. Adult hippocampal neurogenesis is dynamically regulated by physiological and pathological stimuli and believed to be involved in specific brain functions, such as leaming and memory. Defect in adult neurogenesis has also been implicated in certain brain disorders. Adult neurogenesis recapitulates the complete neuronal developmental process in a mature brain environment, including proliferation and fate specification of neural progenitors, neuronal morphogenesis, migration, axon and dendritic development, and synapse development by neuronal progeny. Our recent studies and others showed that neuronal development in the adult brain follows a stereotypic pattern in reaching same milestones as in embryonic neurogenesis, yet the integration process for adult-born neuron is significantly prolonged. Such a stereotypic and prolonged development process for a single neuronal subtype (dentate granule cell) in a relative "steady-state" of mature brain offers a unique model system to investigate mechanisms of neuronal development in vivo in a great detail. We have developed a "single-cell genetic'approach for studying the development of newborn granule cells in vivo using a combination of immunocytochemistry, multi-photon confocal microscopy, electron microscopy and electrophysiology. In the cun-ent project, we aim to examine the role and underiying mechanisms of MeCP2 in postnatal hippocampal neurogenesis in vivo with the following hypothesis: MeCP2 regulates the formation, maturation and maintenance of GABAergic and glutamatergic synapses of new neurons in the adult brain. Our project, addressing in great detail the cell autonomous roles of MeCP2 in vivo, will contribute from a unique aspect to the main goal of the whole center in understanding the molecular basis of RTT. Findings from these studies will be cross-compared with those from the olfactory system (Project 2) to elucidate similarities and differences of neuronal functions of MeCP2 in different developmental stages and brain regions. Random X-inactivation of MECP2 occurs in female and even those with favorable skewing of X inactivation and predominant expression of the WT MECP2 allele exhibit learning disability. Our model system examining individual neurons with MeCP2 dysfuncl^tion in a normal neuronal environment thus have significant clinical implications for the pathophysiology and etiology of RTT. n addition, RTT normally manifests at 6-18 months of age well beyond the primary neurogenesis, our studies of functional roles of MeCP2 in postnatal neurogenesis may thus provide additional novel insights. More importantly, such in vivo system provides a platform for exploring pharmacological and behavioral therapeutic approaches that can be eventually applied in humans to overcome such brain disorder (Project 1), the ultimate goal of the center. RELEVANCE (See instaictions):
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1 |
2014 — 2018 |
Ming, Guo-Li |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Circuitry Mechanisms Underlying Normal and Aberrant Adult Hippocampal Neurogenesis @ Johns Hopkins University
DESCRIPTION (provided by applicant): Adult neurogenesis recapitulates the whole process of neuronal development in a mature central nervous system, from proliferation and fate specification of adult neural progenitors, morphogenesis, migration, axon/dendritic development, and finally synapse formation, culminating in the full integration of new neurons into the existing circuitry. Cumulative evidence suggests that new neurons participate in specific brain functions and aberrant adult neurogenesis may contribute to brain disorders. During the past 11 years, my laboratory has been using adult hippocampal neurogenesis as an experimental model system to elucidate molecular mechanisms regulating the neuronal development. Furthermore, we have been using this system to explore novel functions of risk genes for mental disorders in neuronal development. A number of susceptibility genes for schizophrenia have been identified from human genetic studies. Among them, DISC1 (disrupted-in- schizophrenia 1) was initially identified at the breakpoint of a balanced (1;11)(q42;q14) translocation that co- segregates with schizophrenia in a large Scottish family. Recent studies have further implicated DISC1 as a general risk factor not only for schizophrenia, but also for bipolar disorders and major depression. Functionally, DISC1 is a multifunctional scaffold protein that regulates neuronal development during embryonic, early postnatal and adult neurogenesis. The current project is built upon discoveries made in my laboratory during the past 11 years, both at the cellular lever on sequential phases of new neuron development during adult neurogenesis and critical roles of DISC1 in regulating multiple phases of newborn granule cell development in the adult hippocampus and at the system level for the requirement of DISC1 function specifically in newborn neurons on hippocampal-dependent cognitive and affective behavioral deficits. How genetic dysregulation of DISC1 contributes to aberrant adult neurogenesis and a wide spectrum of mental disorders at the circuitry level is unknown. Our overall goal of this project is to elucidate local internneuron circuitry mechanisms underlying normal neuronal development during adult hippocampal neurogenesis and aberrant development due to DISC1-deficency. Our overall hypothesis is that genetic risk factors for mental disorders interact with specific neurona circuit activity to manifest developmental defects. We are uniquely positioned to address this fundamental questions using genetic, optogenetic, trans-synaptic tracing and imaging tools we have developed. Our proposed studies may not only provide novel mechanistic insights of normal and aberrant neuronal development, but also lead to better understanding of certain mental disorders. One example is our hypothesis-driven identification of epistatic interaction between DISC1 and FEZ1 and between DISC1 and NKCC1 in affecting schizophrenia risks and brain function in humans, all based on our studies from animals using adult neurogenesis as a model system.
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1 |
2015 — 2016 |
Ming, Guo-Li |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Forebrain Organoid Platform For Modelling Human Cortical Neurogenesis @ Johns Hopkins University
? DESCRIPTION (provided by applicant): A major breakthrough in the stem cell field over the last decade has been the development of technology to reprogram human somatic cells into induced pluripotent stem cells or iPSCs. In addition to the ability to derive specific cell types from human iPSCs in dish as 2D cultures, rapid progress in the field has made it possible to generate 3D cultures, or organoids, from iPSCs resembling whole developing organs, including intestinal, kidney, retinal, and cerebral cortex. Human organoids provide a unique opportunity to model organ development in a culture system that is similar to human organogenesis in vivo. Furthermore, organoid cultures provide the opportunity to model diseases that affect multiple cell types and to investigate non-cell- autonomous effects. The work in my laboratory focuses on neural development using both mouse models and iPSC models; and our long term goal is to understand mechanisms underlying normal brain development, neurodevelopmental diseases and to aid in the development of rational therapeutic strategies. Despite the tremendous promise of cerebral organoids to model brain genesis and brain diseases, there are several major limitations of the currently available technology, including high cost, low reproducibility and high variability, that limit our ability for quantitative analyses and broad application of the technology. We have recently developed a new approach by miniaturizing the critical components used to generate cerebral organoids, which allows for a dramatic reduction in materials, cell culture media, space and costs. In this exploratory project, we propose to further standardize forebrain specific organoid production and optimize cell culture conditions for directed and sustained growth. As a proof-of-principle, we will use this system to test the hypothesis that 15q11.2 microdeletion, a prominent genetic risk factor for epilepsy, leads to aberrant cortical neurogenesis for seizure susceptibility. We believe that our approach will transform organogenesis modeling and facilitate the identification of disease-relevant biological processes that are difficult to recapitulate in 2D monolayer cultures.
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1 |
2016 — 2017 |
Ming, Guo-Li |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Deconstructing the Hypothalmic Ontogeny and Plasticity Via Clonal Analysis @ University of Pennsylvania
? DESCRIPTION (provided by applicant): Mammalian brain function critically relies on sophisticated cytoarchitectonic organization during embryonic development. Cell generation, migration, synapse formation and circuit integration often follow highly stereotyped patterns to form complex laminated or nuclear structures in the brain. Recently developed clonal lineage analysis has revealed stem cell behavior giving rise to laminar structures, such as the cerebral cortex, cerebellum and retina, with unprecedented single-cell resolution. However, the formation of nuclear structures by neural stem cells (NSCs) remains unclear and has yet to be systematically investigated. The mammalian hypothalamus is a heterogeneous nuclear structure that is critical for the integration and homeostatic maintenance of endocrine, autonomic and behavioral functions. Reconstructing how hypothalamic neurons are generated from individual NSCs and organized into discrete nuclei during early development is essential to understand the structure-function relationship of different hypothalamic nuclei and the extent to which this can be modulated by environmental conditions. We have developed a genetically-based single-cell lineage tracing-technique that employs MADM (mosaic analysis of double marker) animals to label NSCs in the developing embryo and begin to address these outstanding questions of hypothalamic organization. The goal of the proposed research is to reconstruct and quantify the behavior of individually-labeled NSCs in vivo, decipher the general principles organizing hypothalamic nuclei, decode the ontogeny of individual hypothalamic nuclei and explore the ontogenetic plasticity of nuclear organization in the context of a maternal challenge. The complexity of the anatomical and molecular subdivisions of the hypothalamus, and lack of appropriate genetic tools, has thus far prevented a deep understanding of the organization and ontogeny of this nuclear structure. Successful completion of our study will result in a comprehensive map of single NSCs and their progeny at regional, zonal and nuclear levels, the clonal organization of sibling neurons in a three-dimensional context to determine migratory patterns of newly born neurons, and the capacity of single stem cells to contribute to functionally distinct nuclei. We will also have validated an experimental platform for future mechanistic investigations of hypothalamic dysregulation under pathological conditions, which can lead to targeted diagnostic and therapeutic strategies to preserve critical physiological functions.
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1 |
2016 — 2020 |
Ming, Guo-Li |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Translational Models of Neurodevelopmental Epigenetics and Disease Using Human Ipscs @ University of Pennsylvania
PROJECT ABSTRACT ? Project 3 N/A per PAR-14-183
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1 |
2017 — 2021 |
Ming, Guo-Li |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Administrative Core @ University of Pennsylvania
SUMMARY ? Administrative Core A strong Administrative Core is essential for providing leadership and infrastructure for both scientific and logistical dimensions of large-scale collaborative efforts such as this proposed multi-site research Center. The goal of this Human Tissue Models for Infectious Diseases Cooperative Research Center (HTMID CRC) is to establish a novel 3D human tissue platform to model viral infections of the developing central nervous system. We therefore need to bring together experts from disparate scientific domains and thus, these objectives to facilitate collaborative interactions become even more critical. By providing the investigators with a single point of contact, the Administrative Core will coordinate all activities, including administrative support, financial support, resource sharing and banking, cross-training of personnel, and data management. There are five major goals of the Administrative Core: to facilitate sharing of resources and collaborations among Center investigators (Aim 1); to coordinate external oversight and timely progression toward milestones for each project (Aim 2); to disseminate all results and methodology to the public in a timely manner (Aim 3); to conduct financial and regulatory oversight (Aim 4); and to solicit applications and award Pilot Developmental Research Project grants (Aim 5). The Administrative Core will provide additional support to the Center by keeping team members apprised of the latest technological advances in data collection, storage, sharing, and analysis. The Administrative Core will also take a proactive stance toward coordinating with other Centers to adopt data structures that can facilitate the eventual sharing of data across Centers. Through these activities, the Administrative Core will support individual investigators, research teams, collaborators and consultants to ensure a successful collaborative effort through centralized coordination of the proposed Projects and Cores.
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0.946 |
2017 — 2021 |
Ming, Guo-Li |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Custom-Engineering a 3d Brain Organoid Platform to Model Viral Infections of the Central Nervous System @ University of Pennsylvania
SUMMARY ? Project 1 Despite the tremendous promise of cerebral organoid technology for studying human brain development, modeling brain diseases and testing drug efficacy, we are still at the beginning of an era to engineer mini- brains from human stem cells. For broad applications in basic and translational research, organoid development should be scalable, cost efficient and highly consistent. Previous methods are largely based on cell self-assembly with little external control, and organoids generated by this approach exhibit large variability from sample to sample. We recently developed a miniaturized bioreactor and a protocol to generate forebrain- specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, and gene expression. We employed the forebrain organoid platform to model Zika virus (ZIKV) exposure and showed that preferential, productive infection of neural progenitors by ZIKV leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. In this collaborative research Center, we will investigate the effects of ZIKV and West Nile virus infections at different time points of organoid development to evaluate our platform with two different neurotropic flaviviruses that may target different stages of neural development. Project 1 will focus on technology development to reduce organoid heterogeneity and allow for quantitative analyses of the effects of different viruses on organoid development. To model later stages of brain development, we will adopt state-of-the-art bioengineering approaches to improve the diffusion of media to the organoids and reconstitution of different cell types. And finally, we will develop a medium-throughput platform for compound testing and perform a pilot assay using compounds with known biological effects on viral-infected cells.
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0.946 |
2017 — 2021 |
Ming, Guo-Li Tang, Hengli (co-PI) [⬀] |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Engineering a Human Brain Organoid-Based Platform to Study Neurotropic Viruses @ University of Pennsylvania
SUMMARY ? Overview Modeling of infectious diseases that affect the human central nervous system (CNS), such as those associated with Zika virus (ZIKV) and West Nile virus (WNV), has been challenging due to the inaccessibility of the relevant cell types. Reprogramming human somatic cells, such as skin fibroblasts, into induced pluripotent stem cells (iPSCs) provides a genetically tractable and renewable source of human neural cell populations. We can differentiate these iPSCs into many of the cell types critical for the study of neurotropic viruses, but typically this is performed in monolayer cultures to allow for more control and to generate more homogeneous cell populations, but this methodology lacks the self-organizing properties and interactive dynamics among different cell populations observed during organ development. Recently, more complex structures resembling whole developing organs, named organoids, have been generated from human iPSCs via 3D culturing methods. This emerging new technology has the potential to significantly advance our understanding of infectious diseases and for future therapeutic development. The success of this approach, however, critically depends on how well organoids mimic biological structures, recapitulate human physiology and disease pathology, and incorporate components critical to disease and human host responses. We propose to develop a robust platform for organoid development to model brain development that can be adopted by single labs for basic research, and is amenable to translational studies and drug development and testing. Our Research Center is comprised of three Research Projects, a Scientific Core, and an Administrative Core led by experts in virology, stem cell biology, neural development, and bioengineering. We will focus on ZIKV and WNV, two neurotropic flaviviruses, to develop our organoid platform, which can then be used by the scientific community to investigate other infectious diseases that affect the nervous system. Importantly, ZIKV and WNV are thought to impact the CNS at different stages of development, with ZIKV having been recently implicated as being causal for microcephaly in some pregnant women. This affords us the opportunity to develop an organoid platform with proof-of-principle testing with viruses suspected to have cell type- and stage-specific tropism. Project 1 will focus on technology development to generate more mature organoids and the scaling up of robust assays to perform medium-throughput compound testing. Project 2 will focus on ZIKV infections in early stage organoids and Project 3 will evaluate co-culture organoid systems to model WNV infections in later stage organoids. The projects will be supported by a Scientific Core that will provide cells and on-site training to Projects 2 & 3, as well as optimization of differentiation protocols and bioinformatics analyses. Finally, the Administrative Core will provide logistical support to facilitate collaborations among investigators and to coordinate the timely release of results and resources to the scientific community.
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0.946 |
2017 — 2021 |
Ming, Guo-Li |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Functional Roles of Genetic Risk Factors For Brain Disorders in Neurogenesis and Neurodevelopment @ University of Pennsylvania
The overarching goal of this program is to define cellular and molecular events during neural development vulnerable to genetic perturbations that increase risk for neurodevelopmental and neurological disorders. Currently, our knowledge of human brain development is largely inferred from animal models, indirect measures of human development, and limited access to human neural tissue. All of these are valid tools to piece together the sequential processes of human neural development but are not sufficient to describe the dynamics with enough temporal or molecular resolution to understand mechanistically how genetic risk factors can affect brain formation and function. Technological advances in cellular reprogramming have now made it possible to derive induced pluripotent stem cells (iPSCs) from adult patients, which are a renewable resource for the generation of human neurons with disease-relevant genetic features. This long-term research program is designed to incorporate human iPSC-based studies with animal models to provide a comprehensive and longitudinal understanding of neural development, from neural stem cell behavior to neuronal development, synapse formation and circuit integration. As a proof-of-principle, these studies will use a prominent copy number variation (CNV) risk factor for multiple neurological disorders, 15q11.2CNVs, to illustrate how multifaceted interrogations of the basic biology of neural development in the context of genetic variation can reveal new targets for testing mechanism-based intervention in relevant subtypes of human neurons, as well as animal models of neural function and behavior. Building on significant scientific discoveries we have made in the fields of stem cell biology, adult neurogenesis, and patient-specific iPSCs, and technological innovations we have developed to meet critical challenges in each of these fields, our primary research focus is to integrate multiple levels of analysis to provide a high-resolution description of the cellular processes and molecular mechanisms of neural development that can be used to probe genetic or environmental risk for neurological disorders. Three interlinked projects will be pursued. Project 1 will focus on adult mouse neurogenesis as a model for neural development and use clonal analysis of neural stem cells and their development, single-cell transcriptome analysis, and transgenic mouse models to dissect molecular, cellular, and circuit level effects of genetic mutations on neural development; Project 2 will use human iPSCs with known genetic risk factors, and targeted differentiation protocols, to interrogate human neural development in 2D and 3D cultures; and Project 3 will focus on identifying the molecular mechanisms and targets of risk genes in both animal models and human iPSC-derived neurons and the rescue of observed deficits through rational therapeutic intervention. This is an opportune moment to synthesize recently developed technologies and build a novel translational platform to study underlying mechanisms of neurological disorders, and facilitate the identification of strategies to diagnose, treat, and prevent the often debilitating consequences of dysregulated neural development.
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1 |
2020 |
Ming, Guo-Li |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Sliced Human Neocortical Organoids For Modeling Cortical Laminar and Columnar Organization and Function @ University of Pennsylvania
SUMMARY The modular organization of the cerebral cortex is defined by anatomically and functionally segregated cortical columns, as well as layer-specific anatomical and functional connections that span multiple columns. Dysregulation of the developmental processes governing cortical formation can result in dysmorphic features that have been implicated in numerous neurological and psychiatric disorders. Understanding the basic principles of cortical development has largely relied on animal models but recent advances in 3D organoid cultures using human induced pluripotent stem cells (hiPSCs) have provided unprecedented opportunities to study the intrinsic properties of human neural stem cells and neural progenitors that give rise to highly organized structures in the central nervous system. To date, hiPSC-based cortical organoid models have captured the molecular and cellular dynamics in early stages of fetal human brain development but diffusion limits within the culture system have prevented modeling of later stages of human prenatal and perinatal development. To better model these later stages of human brain development that give rise to laminar and columnar organization, we have developed a sliced organoid culture platform that allows for continuous neurogenesis and the emergence of hallmark features of human cortical anatomy. In this project we will further characterize and validate this strategy (Aim 1) using single-cell RNA-sequencing, immunohistology, electrophysiology and electron microscopy. We will also fuse dorsal and ventral forebrain organoids to allow for the integration of constituent cell types in the cerebral cortex arising from distinct lineages. We will perform anatomical and functional mapping of the circuitry using virus-based trans-synaptic tracing, calcium imaging, and electrophysiology, as well as pharmacological and genetic perturbations to probe the functional implications of laminar (Aim 2) and columnar (Aim 3) organization. Finally, we will perform clonal lineage- tracing to test the hypothesis that functional cortical columns arise from distinct progenitors and radial migration of daughter cells (Aim 3). In sum, these experiments will lead to a human stem cell-based model to understand the human-specific molecular and cellular processes that govern cerebral cortex development and the emergence of functional and anatomical specificity in cortical modules.
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0.946 |