Publications by authors named "Scott A Noggle"

23 Publications

  • Page 1 of 1

Selective linkage of mitochondrial enzymes to intracellular calcium stores differs between human-induced pluripotent stem cells, neural stem cells, and neurons.

J Neurochem 2021 Mar 12;156(6):867-879. Epub 2020 Sep 12.

Burke Neurological Institute, Brain and Mind Research Institute, Weill Cornell Medicine, White Plains, NY, USA.

Mitochondria and releasable endoplasmic reticulum (ER) calcium modulate neuronal calcium signaling, and both change in Alzheimer's disease (AD). The releasable calcium stores in the ER are exaggerated in fibroblasts from AD patients and in multiple models of AD. The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme complex, is diminished in brains from AD patients, and can be plausibly linked to plaques and tangles. Our previous studies in cell lines and mouse neurons demonstrate that reductions in KGDHC increase the ER releasable calcium stores. The goal of these studies was to test whether the relationship was true in human iPSC-derived neurons. Inhibition of KGDHC for one or 24 hr increased the ER releasable calcium store in human neurons by 69% and 144%, respectively. The effect was mitochondrial enzyme specific because inhibiting the pyruvate dehydrogenase complex, another key mitochondrial enzyme complex, diminished the ER releasable calcium stores. The link of KGDHC to ER releasable calcium stores was cell type specific as the interaction was not present in iPSC or neural stem cells. Thus, these studies in human neurons verify a link between KGDHC and releasable ER calcium stores, and support the use of human neurons to examine mechanisms and potential therapies for AD.
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http://dx.doi.org/10.1111/jnc.15160DOI Listing
March 2021

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

Lipid Deprivation Induces a Stable, Naive-to-Primed Intermediate State of Pluripotency in Human PSCs.

Cell Stem Cell 2019 07 30;25(1):120-136.e10. Epub 2019 May 30.

Developmental Biology, The Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-β gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.
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http://dx.doi.org/10.1016/j.stem.2019.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549840PMC
July 2019

Dysregulation of Nutrient Sensing and CLEARance in Presenilin Deficiency.

Cell Rep 2016 Mar 25;14(9):2166-2179. Epub 2016 Feb 25.

Federated Department of Biological Sciences, Rutgers University/New Jersey Institute of Technology, Newark, NJ 07102, USA. Electronic address:

Attenuated auto-lysosomal system has been associated with Alzheimer disease (AD), yet all underlying molecular mechanisms leading to this impairment are unknown. We show that the amino acid sensing of mechanistic target of rapamycin complex 1 (mTORC1) is dysregulated in cells deficient in presenilin, a protein associated with AD. In these cells, mTORC1 is constitutively tethered to lysosomal membranes, unresponsive to starvation, and inhibitory to TFEB-mediated clearance due to a reduction in Sestrin2 expression. Normalization of Sestrin2 levels through overexpression or elevation of nuclear calcium rescued mTORC1 tethering and initiated clearance. While CLEAR network attenuation in vivo results in buildup of amyloid, phospho-Tau, and neurodegeneration, presenilin-knockout fibroblasts and iPSC-derived AD human neurons fail to effectively initiate autophagy. These results propose an altered mechanism for nutrient sensing in presenilin deficiency and underline an importance of clearance pathways in the onset of AD.
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http://dx.doi.org/10.1016/j.celrep.2016.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793148PMC
March 2016

Raising the standards of stem cell line quality.

Nat Cell Biol 2016 Mar;18(3):236-7

New York Stem Cell Foundation, 1995 Broadway, Suite 600, New York, New York 10023, USA.

Yaffe and colleagues discuss the issues surrounding the authentication and quality of induced pluripotent stem cells.
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http://dx.doi.org/10.1038/ncb3313DOI Listing
March 2016

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells.

Nat Methods 2015 Sep 3;12(9):885-92. Epub 2015 Aug 3.

The New York Stem Cell Foundation Research Institute, New York, New York, USA.

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.
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http://dx.doi.org/10.1038/nmeth.3507DOI Listing
September 2015

iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease.

Cell Rep 2014 Nov 6;9(4):1173-82. Epub 2014 Nov 6.

The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA. Electronic address:

Parkinson's disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins' fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of "footprint-free" iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin's neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin.
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http://dx.doi.org/10.1016/j.celrep.2014.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255586PMC
November 2014

Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin.

Hum Mol Genet 2015 Mar 4;24(5):1420-31. Epub 2014 Nov 4.

Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA, Center for Motor Neuron Biology and Disease, Departments of Pathology and Cell Biology, Neurology, and Neuroscience, Columbia Stem Cell Initiative,

Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types.
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http://dx.doi.org/10.1093/hmg/ddu556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402342PMC
March 2015

Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies.

Am J Physiol Cell Physiol 2015 Feb 12;308(3):C209-19. Epub 2014 Nov 12.

New York Stem Cell Foundation Laboratory, New York, New York;

Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation.
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http://dx.doi.org/10.1152/ajpcell.00263.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312839PMC
February 2015

A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs).

PLoS One 2014 29;9(7):e103418. Epub 2014 Jul 29.

Department of Pathology & Cell Biology, Columbia University, New York, New York, United States of America; The Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University, New York, New York, United States of America.

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48-55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103418PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4114788PMC
November 2015

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

PLoS One 2014 8;9(1):e84547. Epub 2014 Jan 8.

The New York Stem Cell Foundation, New York, New York, United States of America.

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084547PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885572PMC
September 2014

Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.

Acta Neuropathol Commun 2014 Jan 7;2. Epub 2014 Jan 7.

The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA.

Background: Induced pluripotent stem cells (iPSCs) derived from patients with neurodegenerative disease generally lack neuropathological confirmation, the gold standard for disease classification and grading of severity. The use of tissue with a definitive neuropathological diagnosis would be an ideal source for iPSCs. The challenge to this approach is that the majority of biobanked brain tissue was not meant for growing live cells, and thus was not frozen in the presence of cryoprotectants such as DMSO.

Results: We report the generation of iPSCs from frozen non-cryoprotected dural tissue stored at -80°C for up to 11 years. This autopsy cohort included subjects with Alzheimer's disease and four other neurodegenerative diseases.

Conclusions: Disease-specific iPSCs can be generated from readily available, archival biobanked tissue. This allows for rapid expansion of generating iPSCs with confirmed pathology as well as allowing access to rare patient variants that have been banked.
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http://dx.doi.org/10.1186/2051-5960-2-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895779PMC
January 2014

Standardization of human stem cell pluripotency using bioinformatics.

Stem Cell Res Ther 2013 Apr 25;4(2):37. Epub 2013 Apr 25.

The study of cell differentiation, embryonic development, and personalized regenerative medicine are all possible through the use of human stem cells. The propensity for these cells to differentiate into all three germ layers of the body with the potential to generate any cell type opens a number of promising avenues for studying human development and disease. One major hurdle to the development of high-throughput production of human stem cells for use in regenerative medicine has been standardization of pluripotency assays. In this review we discuss technologies currently being deployed to produce standardized, high-quality stem cells that can be scaled for high-throughput derivation and screening in regenerative medicine applications. We focus on assays for pluripotency using bioinformatics and gene expression profiling. We review a number of approaches that promise to improve unbiased prediction of utility of both human induced pluripotent stem cells and embryonic stem cells.
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http://dx.doi.org/10.1186/scrt185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707009PMC
April 2013

Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.

PLoS One 2013 29;8(3):e59867. Epub 2013 Mar 29.

The New York Stem Cell Foundation, New York, New York, United States of America.

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13(NEG)SSEA4(POS)Tra-1-60(POS) on day 7-10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and "contaminating" partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612089PMC
January 2014

Differentiation of serum-free embryoid bodies from human induced pluripotent stem cells into networks.

Stem Cell Res 2013 May 13;10(3):454-63. Epub 2013 Feb 13.

The New York Stem Cell Foundation Laboratory 178 Columbus Avenue #237064 New York, NY 10023, USA.

Three-dimensional aggregation cultures allow for complex development of differentiated human induced pluripotent stem cells. However, this approach is not easily amenable to live-cell imaging and electrophysiological applications due to the thickness and the geometry of the tissue. Here, we present an improvement on the traditional aggregation method by combining the use of cell culture inserts with serum-free embryoid bodies (SFEBs). The use of this technique allows the structures to maintain their three-dimensional structure while thinning substantially. We demonstrate that this technique can be used for electrophysiological recodings as well as live-cell calcium imaging combined with electrical stimulation, akin to organotypic slice preparations. This provides an important experimental tool that can be used to bridge 3-D structures with traditional monolayer approaches used in stem cell applications.
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http://dx.doi.org/10.1016/j.scr.2013.02.001DOI Listing
May 2013

Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants.

Nature 2013 Jan 19;493(7434):632-7. Epub 2012 Dec 19.

The New York Stem Cell Foundation Laboratory, New York, NY 10032, USA.

Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. Here we explore the use of nuclear genome transfer between unfertilized oocytes of two donors to prevent the transmission of mitochondrial mutations. Nuclear genome transfer did not reduce developmental efficiency to the blastocyst stage, and genome integrity was maintained provided that spontaneous oocyte activation was avoided through the transfer of incompletely assembled spindle-chromosome complexes. Mitochondrial DNA transferred with the nuclear genome was initially detected at levels below 1%, decreasing in blastocysts and stem-cell lines to undetectable levels, and remained undetectable after passaging for more than one year, clonal expansion, differentiation into neurons, cardiomyocytes or β-cells, and after cellular reprogramming. Stem cells and differentiated cells had mitochondrial respiratory chain enzyme activities and oxygen consumption rates indistinguishable from controls. These results demonstrate the potential of nuclear genome transfer to prevent the transmission of mitochondrial disorders in humans.
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http://dx.doi.org/10.1038/nature11800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924261PMC
January 2013

A molecular basis for human embryonic stem cell pluripotency.

Stem Cell Rev 2005 ;1(2):111-8

Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, NY 10021-6399, USA.

Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFbeta, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFbeta signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.
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http://dx.doi.org/10.1385/SCR:1:2:111DOI Listing
January 2007

Contribution of human embryonic stem cells to mouse blastocysts.

Dev Biol 2006 Jul;295(1):90-102

Laboratory of Molecular Embryology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

In addition to their potential for cell-based therapies in the treatment of disease and injury, the broad developmental capacity of human embryonic stem cells (hESCs) offers potential for studying the origins of all human cell types. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or in teratomas, yet these methods have stopped short of demonstrating the ESC potential exhibited in the mouse (mESCs), which can give rise to every cell type when combined with blastocysts. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. Our results show that hESCs can engraft into mouse blastocysts, where they proliferate and differentiate in vitro and persist in mouse/human embryonic chimeras that implant and develop in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation.
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http://dx.doi.org/10.1016/j.ydbio.2006.03.026DOI Listing
July 2006

Notch signaling is inactive but inducible in human embryonic stem cells.

Stem Cells 2006 Jul 13;24(7):1646-53. Epub 2006 Apr 13.

Department of Genetics, University of Georgia, Davison Life Sciences Complex, Athens, 30602, USA.

The NOTCH signaling pathway performs a wide range of critical functions in a number of different cell types during development and differentiation. The role of NOTCH signals in human embryonic stem cells (hESCs) has not been tested. We measured the activity of canonical NOTCH signaling in undifferentiated embryonic stem (ES) cells and tested the requirement for NOTCH activity in hESC self-renewal or differentiation by growing hESCs in the presence of gamma-secretase inhibitors. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of the differentiating cell types that accumulate in human ES cell cultures. Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors.
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http://dx.doi.org/10.1634/stemcells.2005-0314DOI Listing
July 2006

The mouse vesicular inhibitory amino acid transporter gene: expression during embryogenesis, analysis of its core promoter in neural stem cells and a reconsideration of its alternate splicing.

Gene 2005 May 9;351:39-49. Epub 2005 Apr 9.

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, 30912, USA.

The vesicular inhibitory amino acid transporter, VIAAT (also known as vesicular GABA transporter VGAT) transports GABA or glycine into synaptic vesicles. To initiate an analysis of the expression and regulation of VIAAT during neurogenesis we have cloned and characterized the mouse Viaat gene. We find that the mouse Viaat coding sequence is encoded by two exons spanning 5.3 kb. A survey of expression by whole mount in situ hybridization of mouse embryos indicates that Viaat is activated early in neuron differentiation and is expressed widely within the developing CNS; however, we did not detect expression in the superficial non-neural structures that express the GABA synthase Gad1. Analysis of the Viaat promoter indicates that a minimal promoter region containing a CG rich sequence is sufficient for efficient expression in neural stem and precursor cells. Our analysis of the Viaat sequence and splicing does not support the existence of two Viaat isoforms as previously proposed [Ebihara et al., Brain Res. Mol Brain Res. 110 (2003), 126-139]. Instead, the alternative isoform Viaat-a appears to be due to PCR artifacts that have occurred independently in multiple labs.
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http://dx.doi.org/10.1016/j.gene.2005.01.009DOI Listing
May 2005

Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

Stem Cells 2004 ;22(7):1218-38

BresaGen Inc., 111 Riverbend Rd., Athens, Georgia, 30605, USA.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.
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http://dx.doi.org/10.1634/stemcells.2004-0114DOI Listing
May 2005

Directed neuronal differentiation of human embryonic stem cells.

BMC Neurosci 2003 Oct 22;4:27. Epub 2003 Oct 22.

Department of Animal and Dairy Science, University of Georgia, Athens, 30605, USA.

Background: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).

Results: A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.

Conclusion: This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
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http://dx.doi.org/10.1186/1471-2202-4-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC272931PMC
October 2003

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons.

Biochem Biophys Res Commun 2003 Jun;306(1):191-7

Department of Biochemistry and Molecular Biology, University of Georgia, 111 Riverbend Road, Athens, GA 30605, USA.

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.
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http://dx.doi.org/10.1016/s0006-291x(03)00937-9DOI Listing
June 2003