Su-Chun Zhang - US grants

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells are non-transformed primitive cells derived from early embryos and are capable of essentially unlimited proliferation in an undifferentiated state yet retain the potential to give rise to all cell and tissue types of the body. Thus ES cells provide an almost limitless source for deriving specialized cells for cell therapy. Both human and nonhuman primate ES cell lines have been established by James Thomson of the Wisconsin Regional Primate Research Center and they behave similarly in vitro and in vivo. This proposal will explore the feasibility of using rhesus monkey ES cells (which are available only from the Wisconsin Regional Primate Research Center) as a source to produce dopamine neurons, the major cell type lost in human Parkinson?s disease. First, the ES cells will be directed to neural precursors in a "neural promoting" condition. Second, the ES cell-derived neural precursors will be coaxed to become specialized dopamine neurons based on the developmental requirement of midbrain dopamine neurons. Third, the ES cell-derived dopamine neurons will be examined for their therapeutic potential in restoring motor dysfunction in a rat model of Parkinson?s disease. If successful, this exploratory/developmental grant (R21) will be expanded to a five-year proposal to transplant the monkey ES cell-derived dopamine neurons to a monkey model of Parkinson?s disease in the future. Information gained from these studies will be crucial in ultimately utilizing human ES cells to treat neurological illnesses including Parkinson?s disease.

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs) can be directed to multiple cell lineages including the neural lineage. They thus offer a unique model to reveal molecular interactions underlying early neural specification that occurs in an experimentally inaccessible early human embryo. In our previous R01 project, we discovered, unexpectedly, that the transcription factor Pax6 is expressed uniformly and specifically in early or primitive neuroepithelial (NE) cells that are differentiated from hESCs. This is reminiscent of that of Sox1, the earliest and definitive NE transcription factor in vertebrate animals. We now show that forced expression of Pax6 results in neuroepithelial differentiation in human but not mouse ESCs whereas inhibition of Pax6 expression suppresses neuroepithelial differentiation from human but not mouse ESCs. We therefore hypothesize that Pax6 acts as a key mediator of the NE fate of hESCs and that control of human NE specification may be achieved through regulation of Pax6 expression. We will build hESC lines that conditionally express Pax6 or Sox1 and inhibit Pax6 or Sox1 expression via RNA interference (RNAi). Differentiation of these transgenic hESCs to NE using our defined system will address whether Pax6 is necessary and/or sufficient for NE specification from hESCs. This will reveal a novel role of the conserved protein Pax6 in human NE specification. We will then identify extracellular factors that regulate NE specification and determine if these factors do so by directly regulating Pax6 expression using chromatin immunoprecipitation and DNA binding assays. This will directly link extracellular factors to neuroectoderm transcription factors for the first time. Finally, we will assess whether NE specification from hESCs, especially the maintenance of the primitive state of NE and hence the plasticity to be re-patterned may be controlled by regulating Pax6 expression using our transgenic Pax6 hESC lines. If so, we will uncover a way to maintain somatic (neural) stem cells. Together, information gained from this proposal will provide us with a means of controlling the fate choice of human stem cells. It will also be instrumental to expanding the repair potential of the stem cells that are present in our brain.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from the progressive loss of dopamine (DA) neurons in the midbrain. Replacement of the lost DA neurons with fetal midbrain cells through neural transplantation in clinical trials has produced clinical benefits and has laid a foundation for cell therapy in PD. This therapy, however, is hindered by the limited supply of effective donor cells. Human embryonic stem (hES) cells (NIH Registry WA01 and WA09), established from the inner cell mass of a preimplantation embryo, are capable of almost unlimited proliferation in an undifferentiated state, yet retain the potential to differentiate into almost all cell and tissue types of the body including DA neurons. Thus ES cells may provide a simple and continual source of specialized human cells, which can be standardized and banked. This application is to resolve a single but crucial issue surrounding potential stem cell replacement therapy for PD, i.e., which hES-derived cell type, neuroepithelial cells, DA neuron progenitors, or DA neurons, is best for transplant therapy in PD. This study is based on our success in guiding hES cells to neuroepithelial cells, DA neuron progenitors and mature DA neurons in culture. The criteria for determining the candidate cell type include safety to recipients, efficacy of the cells for functional replacement, efficiency in cell production, and simplicity for standardization of cell preparation procedures. The proposed study will determine the ideal hES-derived cells for PD therapy, thus leading to preclinical studies to transplant the selected cells into a monkey PD model we have established, and to bank and/or standardize the cell production in our Biomanufacturing Facility before clinical trials.

stem cells; dopamine; neurophysiology; neurons; Macaca mulatta; animal colony;

[unreadable] DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs), like their mouse counterparts, provide a useful tool to unveil cellular and molecular events underlying normal and abnormal human development. Directed differentiation of hESCs may allow establishing systems for pharmaceutical screening and producing functional cells/tissues for potential regenerative medicine. Unlike their mouse counterparts, hESCs are difficult to modify genetically. Technical difficulties in genetically modifying the slowly growing hESCs have become a significant barrier for most laboratories to proceed with their individual research programs using hESCs. We propose to engineer a common hESC line with the Cre-recombination mediated exchange system for conditional gene expression and knockdown. This proposal is based on our success in building a constitutive master hESC line that carries an exchangeable cassette. We will first modify the constitutive master hESC line (derived from NIH Registry WA09 and WA01) by using an inducible vector through the same Cre recombination mediated mechanism to build master inducible hESC lines with exchangeable cassette for transgene expression or knockdown (Aim 1). We will then build hESC lines to conditionally express a neurogenic gene neurogenin 2 (Ngn2) and knock down a pluripotent gene Oct4 through Cre mediated recombination at the unique loxP sites, and confirm that the target gene expression can be regulated in vitro and following transplantation into the immune deficient mice (Aim 2). These common hESC lines will be deposited in the NIH-sponsored National Stem Cell Bank. Availability of such a conditional master hESC line will provide a flexible and ease platform to manipulate hESCs in most laboratories for both basic and applied researches. It will ultimately speed up the potential use of hESCs in regenerative medicine.. [unreadable] [unreadable] [unreadable]

The ultimate goal of this project is to explore cellular and molecular replacement strategies for treating motor neuron degeneration. Our approach entails comprehensive analyses of both intrinsic properties and extrinsic factors that affect the survival of human motor neurons in adult almyotrophic lateral sclerosis (ALS) spinal cord. Our logic is based on the following premises: (1) Motor neuron degeneration in ALS may not be cell autonomous to motor neurons but may arise, in part, from an abnormal glial environment. Thus, modifying the glial environment will protect endogenous motor neurons and create a healthy milieu for grafted motor neurons. (2) We have established a system to efficiently produce healthy human glial cells, such as astrocytes, as well as motor neurons from naive embryonic stem cells (ESCs). These neural cells may represent a source of therapeutic agents, as well as a tool for dissecting neural cell interactions during motor neuron degeneration. (3) Human stem cells, including ESCs and neural stem/progenitor cells, can be genetically modified to carry therapeutic genes. This project will take advantage of advances in our understanding of intermittent hypoxia-induced respiratory motor neuron plasticity under investigation in Project 1, and build upon the foundation of motor neuron-astrocyte interactions in ALS pathogenesis assessed in Project 2. We will genetically modify hESCs (NIH Registry, WA01, WA09) to carry cell death-resistant genes and transplant the differentiated human motor neurons into the spinal cord of ALS rats. Through interactions with projects 1 & 2, we will evaluate whether and how intermittent hypoxia, a respiratory exercise, and growth factor-producing astrocytes promote the survival, integration, and axonal growth of the grafted human motor neurons. Our goal is to identify an ideal combinatory strategy to achieve successful transplantation of human motor neurons in the adult ALS spinal cord. Together with projects 1 & 2, we hope to arrive at a comprehensive strategy for treating the devastating ALS.

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease caused by mutations in the "survival of motor neuron (SMN)" gene, leading to spinal motor neuron degeneration and muscular atrophy. It is one of the leading causes of infant death without an effective treatment. This pilot proposal intends to create a cellular model of SMA using SMA patient's skin cells. Specifically, we will first induce pluripotent stem (iPS) cells from an SMA patient's fibroblasts by expressing pluripotent transcription factors using a recently established method. We will then direct the SMA-iPS cells, alongside non-SMA iPS cells and human embryonic stem cells (NIH Registry: WA07 and WA09), toward spinal motor neurons using our reproducible differentiation protocol established for human embryonic stem cells. We will determine if the development (differentiation, neurite extension) and function (axonal transportation, interactions with muscles) of motor neurons derived from the SMA-iPS cells are defective. Thus, we will create for the first time SMA-specific human iPS cells, which will permit studies on the pathogenesis of spinal motor neuron degeneration under the human genetic background. This system can also serve as a simple but bona fide human SMA target for therapeutic screening, our main goal. PUBLIC HEALTH RELEVANCE: We are building stem cells from the skin cells of patients with spinal muscular atrophy (SMA). The stem cells will offer a model system to study the pathogenesis of spinal motor neuron degeneration under the human genetic background. It can also serve as a simple but bona fide human SMA target for therapeutic screening, which may ultimately lead to the discovery of a treatment for SMA.

DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from degeneration of midbrain dopamine (DA) neurons and can be effectively treated with L-dopa in the initial phase. However, DA supplementation does not halt the DA neuron degeneration process, nor does it correct the loss of DA neurons. Consequently, PD patients almost invariably lose responsiveness to L-dopa treatment over time. Transplantation of human fetal mesencephalic tissues to replace the lost DA neurons has shown efficacy in alleviating symptoms of some PD patients. This therapy, however, depends on collection of tissues from multiple fetuses of particular ages for a single patient, which makes it impractical for general application and is ethically problematic. This proposal explores the possibility of future personalized cell therapy for PD using a non-human primate model. We will derive safe and functional DA neurons from the skin tissue of individual Parkinsonian rhesus monkeys through generation of induced pluripotent stem cells (iPSCs) that are free of virus and transgenes and using our newly developed strategy for midbrain DA neuron differentiation. We will then label the cell genetically and transplant the midbrain DA neurons back to the monkey from which the cells are derived, and assess whether the DA neurons survive and contribute to therapy in a short term and whether the therapeutic outcome is sustained over a long term (2-3 years). Results from this study will determine the safety and efficacy of autologous stem cell therapy for PD in primates, thus setting up a foundation for future clinical trials using reprogrammed human cells.

Amygdala, especially the lateral central nucleus (CeL), plays a critical role in mediating anxious temperament (AT). However, the cellular, molecular, and functional properties of CeL neurons that underlie AT pathogenesis remain to be explored. The Cellular Neurobiology Core will provide standard technical services to characterize the gene (protein) expression in CeL neurons at the light and electron microscopic levels in qualitative and quantitative manners. We will reprogram induced pluripotent stem cells (iPSCs) from skin biopsy tissues of monkey and human subjects with high and low AT using the state-of-the-art nonintegration technology. These IPSCs will be directed to neuronal subtypes, including the DARPP32- expressing medium spiny GABA neurons that resemble those in the CeL using our established protocols. The gene expression, transmitter release and electrophysiological properties of these neurons will be evaluated by immunocytochemistry, HPLC, and whole cell patch clamping analysis. These cellular properties will be cross verified between cultured neurons and those in the monkey brain and compared between high and low AT. By providing these services and close collaboration with individual projects, the core will generate iPSCs from high and low AT monkeys and human individuals for the first time. These cell lines will become valuable resources for and be made available to investigators across the Conte centers. Information gained from the cellular analyses will enable the discovery of cellular/ functional substrate that underlies AT development and bridge the monkey model to human disorders.

DESCRIPTION (provided by applicant): Astrocytes, the most abundant cell type in the human brain, participate in virtually every aspect of brain function and often disease progression. Defining the roles of astrocytes in normal and abnormal brain development as well as disease pathogenesis has been significantly hampered by the lack / scarcity of markers that are specific to progenitors and more mature cells of the astroglial lineage as well as those that may signal functional diversity of astrocytes in different brain regions. During our differentiation of human pluripotent stem cells (hPSCs) to neural subtypes, we discovered a glial precursor that generates astrocytes but not neurons and oligodendrocytes. We will use these putative astroglial progenitors to discover markers that are specific to astroglial progenitors, thus enabling prospective identification of astroglial progenitors for the first time. Building upon our decade's experience in directing PSCs to region-specific neuronal subtypes, we have successfully generated enriched populations of region-specific astrocyte subtypes. We will use these astrocyte subtypes to uncover the functional characteristics of regional astrocytes, setting up the foundation for further exploring the effects of astrocytes on neuronal function in particula brain regions under homeostatic and pathological conditions. As a proof-of-principle, we will profile the transcriptome of cortical astrocytes from Fragile X Syndrome (FXS) in which dysfunctional astrocytes may play a role and for which we have established iPSCs, and discern potential functional contribution of astrocytes to neuronal dysfunction that underlies FXS. Along the proposed study, we will establish tools and resources (e.g., transcription profiles, reporter/transgenic lines for astroglial cells) that will enhance studies on astroglial lineage development and their contribution to pathogenesis, and enable therapeutic development for mental disorders by targeting astroglial cells.

ABSTRACT Alexander disease (AxD) is a primary disease of astrocytes caused by mutations in the gfap gene. How AxD mutations lead to protein aggregation and astrocyte dysfunction as well as how mutant GFAP-expressing astrocytes result in neuronal degeneration remain unknown. Evolutionarily, astrocytes play increasingly more important and complex roles in human brain functions. Hence, the ability to directly examine AxD patients' astrocytes will not only complement the existing models but also reveal potential unique aspects of human astrocytes in AxD pathogenesis. We have built induced pluripotent stem cells (iPSCs) from AxD patients with three different mutations through close collaboration with the Messing lab (project 3). We have also developed a reproducible strategy to guide hPSCs to enriched astrocytes. Our preliminary study revealed the presence of RFs in AxD patients' astrocytes in culture and following transplantation into the mouse brain, highlighting the recapitulation of key AxD pathology in our iPSC system. However, astrocytes derived from AxD patients exhibit comparable GFAP levels as non-AxD individuals, suggesting that GFAP may not increase at early stages of AxD and a simple increase in GFAP protein might not be the major trigger for RF formation and astrocyte dysfunction in human cells as proposed for model animals. Our ability to produce enriched astrocytes from AxD patients has also led to the discovery that AxD astrocytes have altered ratio of GFAP-d/a isoforms, which has led to the ?rediscovery? of a similar ratio change in transgenic mice. We will use advanced gene editing technology to determine if change of GFAP isoforms mediates the effects of gfap mutations on protein aggregation and/or astrocyte dysfunction. By neuron-astrocyte co-culture and neural transplantation, we will determine if and how AxD astrocytes cause neuronal degeneration. As part of the program project, we will validate modifiers of AxD learned from animal models in our AxD patients' astrocytes/neurons, setting up the foundation for future translation.

DESCRIPTION (provided by applicant): Human Pluripotent Stem cells (hPSCs), including embryonic stem cells (ESCs) and especially, induced pluripotent stem cells (iPSCs) established from patient's tissues, offer a new research tool for understanding disease pathogenesis, a versatile template for drug discovery, and a promising source of cell therapy. A key step in realizing these potentials of human PSCs is genetic modification / gene targeting, as in the creation of transgenic animals that has revolutionized biomedical research and discovery. Unlike their mouse counterparts, transgenesis of hPSCs is inefficient and the transgene expression is often not stable, especially upon neural differentiation, let alone functional regulation. Technical difficulties in genetically modifying hPSCs and unavailability of commonly needed hPSC lines, as opposed to readily available transgenic animals, have become a significant barrier to neuroscience research. The present proposal aims to overcome these hurdles by building and distributing transgenic hPSCs and related reagents. Building upon our decade's experience in hPSC research, we will first establish hESC lines with a (GFP) reporter in neural genes that are most frequently sought by investigators in neuroscience using the TALEN- or CRISPR/CAS-mediated homologous recombination technology (Aim 1). We will also build hPSC lines with a functional regulator in an envy site under the control of a neural promoter (Aim 2), which will allow interrogation of the function of human neural cells in vitro and in vivo. These cell lines will be distributed through the WiCell Institute or an NIH designated distribution center (e.g., Rutgers University Cell and DNA Repository [RUCDR]). To promote neuroscience research in a broad spectrum as well as that involving specialized model systems (e.g., disease iPSCs), we will further deposit the transgenic reagents that are proven effective through Addegene for dissemination (Aim 3). Availability of such hPSC lines and tools will substantially speed up the next wave of neuroscience discovery in a cost effective manner and enable translation to clinical neuroscience.

ABSTRACT Cell therapy holds promise for CNS disorders. This is suggested by clinical benefits in some Parkinson's disease (PD) patients who received transplantation of human fetal mesencephalic tissues to replace degenerated midbrain dopamine (mDA) neurons. The ability to produce functional neurons efficiently from human pluripotent stem cells (PSCs) expands the prospect of cell therapy. However, cell therapy for many neurological conditions faces major hurdles. In particular, transplanted neurons often fail to reconstruct a functional circuit that is specific to lost function. In PD, DA neurons are often transplanted directly into the striatum, rather than their home location substantial nigra. Consequently, the grafted cells and their repaired circuit lose appropriate inputs for functional modulation. To address these critical issues, we will reconstruct the nigra-striatal circuit by cell transplantation to the nigra in a PD model mouse and determine if the grafted human mDA neurons project specifically to the striatum to form functional circuit and if the reconnection is sped up by regulating graft activity (Aim 1). Furthermore, we will explore to regulate the neural circuit repaired by transplanted cells by expressing active or inhibitory form of DREADD (designer receptor exclusively activated by designer drug) in hPSCs so that the therapeutic outcomes may be refined (Aim 2). Information gained from this study will be instrumental for restoring and regulating neural circuitry in PD and other neurological conditions.

ABSTRACT Down syndrome (DS, trisomy 21, T21), a complex multigene disorder and the most common genetic cause of intellectual disability. However, surprisingly little is known about the underlying mechanisms that lead to cognitive impairment in DS. There are fewer neurons in adult DS cortex and reduced neurogenesis and synaptogenesis have been implicated as features of DS development. Yet, what and how specific neurons and synaptic contacts are affected at which period of development and what molecular pathways underlie these defects that lead to intellectual disability remain unclear. We propose to build models based on human induced pluripotent stem cells (iPSCs), to interrogate how T21 disrupts developmental processes in DS. To ensure the validity of the stem cell based models, we will first establish a cellular, synaptic, and molecular atlas of the DS prenatal cortex. By integrating molecular signatures of single cells with the cellular changes both in vivo and in in vitro models, we will tease out the molecular pathways that are disrupted by T21 that account for the altered neural development. The results from these experiments will provide mechanistic understanding of intellectual disability in DS. More broadly, the results will address gaps in our understanding of human cortical neuron development and consequences of mistakes.