Publications by authors named "Su-Chun Zhang"

106 Publications

Human spinal GABA neurons alleviate spasticity and improve locomotion in rats with spinal cord injury.

Cell Rep 2021 Mar;34(12):108889

Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Electronic address:

Spinal cord injury (SCI) often results in spasticity. There is currently no effective therapy for spasticity. Here, we describe a method to efficiently differentiate human pluripotent stem cells from spinal GABA neurons. After transplantation into the injured rat spinal cord, the DREADD (designer receptors exclusively activated by designer drug)-expressing spinal progenitors differentiate into GABA neurons, mitigating spasticity-like response of the rat hindlimbs and locomotion deficits in 3 months. Administering clozapine-N-oxide, which activates the grafted GABA neurons, further alleviates spasticity-like response, suggesting an integration of grafted GABA neurons into the local neural circuit. These results highlight the therapeutic potential of the spinal GABA neurons for SCI.
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http://dx.doi.org/10.1016/j.celrep.2021.108889DOI Listing
March 2021

Autologous transplant therapy alleviates motor and depressive behaviors in parkinsonian monkeys.

Nat Med 2021 04 1;27(4):632-639. Epub 2021 Mar 1.

Waisman Center, University of Wisconsin-Madison, Madison, WI, USA.

Degeneration of dopamine (DA) neurons in the midbrain underlies the pathogenesis of Parkinson's disease (PD). Supplement of DA via L-DOPA alleviates motor symptoms but does not prevent the progressive loss of DA neurons. A large body of experimental studies, including those in nonhuman primates, demonstrates that transplantation of fetal mesencephalic tissues improves motor symptoms in animals, which culminated in open-label and double-blinded clinical trials of fetal tissue transplantation for PD. Unfortunately, the outcomes are mixed, primarily due to the undefined and unstandardized donor tissues. Generation of induced pluripotent stem cells enables standardized and autologous transplantation therapy for PD. However, its efficacy, especially in primates, remains unclear. Here we show that over a 2-year period without immunosuppression, PD monkeys receiving autologous, but not allogenic, transplantation exhibited recovery from motor and depressive signs. These behavioral improvements were accompanied by robust grafts with extensive DA neuron axon growth as well as strong DA activity in positron emission tomography (PET). Mathematical modeling reveals correlations between the number of surviving DA neurons with PET signal intensity and behavior recovery regardless autologous or allogeneic transplant, suggesting a predictive power of PET and motor behaviors for surviving DA neuron number.
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http://dx.doi.org/10.1038/s41591-021-01257-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198752PMC
April 2021

Impaired lipid metabolism in astrocytes underlies degeneration of cortical projection neurons in hereditary spastic paraplegia.

Acta Neuropathol Commun 2020 12 7;8(1):214. Epub 2020 Dec 7.

Department of Biomedical Sciences, University of Illinois College of Medicine Rockford, Rockford, IL, 61107, USA.

Hereditary spastic paraplegias (HSPs) are caused by a length-dependent axonopathy of long corticospinal neurons, but how axons of these cortical projection neurons (PNs) degenerate remains elusive. We generated isogenic human pluripotent stem cell (hPSC) lines for two ATL1 missense mutations associated with SPG3A, the most common early-onset autosomal dominant HSP. In hPSC-derived cortical PNs, ATL1 mutations resulted in reduced axonal outgrowth, impaired axonal transport, and accumulated axonal swellings, recapitulating disease-specific phenotypes. Importantly, ATL1 mutations dysregulated proteolipid gene expression, reduced lipid droplet size in astrocytes, and unexpectedly disrupted cholesterol transfer from glia to neurons, leading to cholesterol deficiency in SPG3A cortical PNs. Applying cholesterol or conditioned medium from control astrocytes, a major source of cholesterol in the brain, rescued aberrant axonal transport and swellings in SPG3A cortical PNs. Furthermore, treatment with the NR1H2 agonist GW3965 corrected lipid droplet defects in SPG3A astrocytes and promoted cholesterol efflux from astrocytes, leading to restoration of cholesterol levels and rescue of axonal degeneration in SPG3A cortical PNs. These results reveal a non-cell autonomous mechanism underlying axonal degeneration of cortical PNs mediated by impaired cholesterol homeostasis in glia.
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http://dx.doi.org/10.1186/s40478-020-01088-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720406PMC
December 2020

Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function.

Cell Stem Cell 2021 01 22;28(1):112-126.e6. Epub 2020 Sep 22.

Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Program in Neuroscience & Behavioral Disorders, Duke-NUS Medical School, 169857 Singapore, Singapore.

Although cell transplantation can rescue motor defects in Parkinson's disease (PD) models, whether and how grafts functionally repair damaged neural circuitry in the adult brain is not known. We transplanted hESC-derived midbrain dopamine (mDA) or cortical glutamate neurons into the substantia nigra or striatum of a mouse PD model and found extensive graft integration with host circuitry. Axonal pathfinding toward the dorsal striatum was determined by the identity of the grafted neurons, and anatomical presynaptic inputs were largely dependent on graft location, whereas inhibitory versus excitatory input was dictated by the identity of grafted neurons. hESC-derived mDA neurons display A9 characteristics and restore functionality of the reconstructed nigrostriatal circuit to mediate improvements in motor function. These results indicate similarity in cell-type-specific pre- and post-synaptic integration between transplant-reconstructed circuit and endogenous neural networks, highlighting the capacity of hPSC-derived neuron subtypes for specific circuit repair and functional restoration in the adult brain.
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http://dx.doi.org/10.1016/j.stem.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796915PMC
January 2021

[F]FEPPA PET imaging for monitoring CD68-positive microglia/macrophage neuroinflammation in nonhuman primates.

EJNMMI Res 2020 Aug 6;10(1):93. Epub 2020 Aug 6.

Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA.

Purpose: The aim of this study was to examine whether the translocator protein 18-kDa (TSPO) PET ligand [F]FEPPA has the sensitivity for detecting changes in CD68-positive microglial/macrophage activation in hemiparkinsonian rhesus macaques treated with allogeneic grafts of induced pluripotent stem cell-derived midbrain dopaminergic neurons (iPSC-mDA).

Methods: In vivo positron emission tomography (PET) imaging with [F]FEPPA was used in conjunction with postmortem CD68 immunostaining to evaluate neuroinflammation in the brains of hemiparkinsonian rhesus macaques (n = 6) that received allogeneic iPSC-mDA grafts in the putamen ipsilateral to MPTP administration.

Results: Based on assessment of radiotracer uptake and confirmed by visual inspection of the imaging data, nonhuman primates with allogeneic grafts showed increased [F]FEPPA binding at the graft sites relative to the contralateral putamen. From PET asymmetry analysis of the images, the mean asymmetry index of the monkeys was AI = - 0.085 ± 0.018. Evaluation and scoring of CD68 immunoreactivity by an investigator blind to the treatment identified significantly more neuroinflammation in the grafted areas of the putamen compared to the contralateral putamen (p = 0.0004). [F]FEPPA PET AI showed a positive correlation with CD68 immunoreactivity AI ratings in the monkeys (Spearman's ρ = 0.94; p = 0.005).

Conclusion: These findings reveal that [F]FEPPA PET is an effective marker for detecting increased CD68-positive microglial/macrophage activation and demonstrates sufficient sensitivity to detect changes in neuroinflammation in vivo following allogeneic cell engraftment.
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http://dx.doi.org/10.1186/s13550-020-00683-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410886PMC
August 2020

PAX6D instructs neural retinal specification from human embryonic stem cell-derived neuroectoderm.

EMBO Rep 2020 09 23;21(9):e50000. Epub 2020 Jul 23.

Waisman Center, University of Wisconsin-Madison, Madison, WI, USA.

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired-less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification. Induced expression of PAX6D but not PAX6A in a PAX6-null background restored the NR specification capacity. ChIP-Seq, confirmed by functional assays, revealed a set of retinal genes and non-retinal neural genes that are potential targets of PAX6D, including WNT8B. Inhibition of WNTs or knocking down of WNT8B restored the NR specification capacity of neuroepithelia with PAX6D knockout, whereas activation of WNTs blocked NR specification even when PAX6D was induced. Thus, PAX6D specifies neuroepithelia to NR cells via the regulation of WNT8B.
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http://dx.doi.org/10.15252/embr.202050000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507545PMC
September 2020

Plasticity of Synaptic Transmission in Human Stem Cell-Derived Neural Networks.

iScience 2020 Feb 9;23(2):100829. Epub 2020 Jan 9.

Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; Program in Neuroscience & Behavioral Disorders, Duke-NUS Medical School, Singapore, Singapore. Electronic address:

Long-term potentiation and depression, inferred from analysis on brain slices, are considered the cellular processes underlying learning and memory formation. They have not so far been demonstrated in human stem cell-derived neurons. By expressing channelrhodopsin in hESCs-derived glutamate neurons and co-culturing them with GABA neurons, we found that blue light stimulation increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and decreased the ratio of paired pulse facilitation (PPF) in non-ChR2-expressing GABA neurons, indicating a facilitating action at the presynaptic terminals. When paired with postsynaptic depolarization, the repetitive stimulation significantly increased the amplitude of light-evoked EPSCs that persisted during the period, indicating long-term potentiation (LTP). In contrast, low-frequency light stimulation induced long-term depression (LTD). These effects were blocked by N-methyl-D-aspartic acid (NMDA) receptor antagonists, suggesting NMDA receptor-mediated synaptic plasticity in human neural networks. Furthermore, induced pluripotent stem cell (iPSC)-derived neurons of patient with Down syndrome showed absence of LTP or LTD. Thus, our platform offers a versatile model for assessing human neural plasticity under physiological and pathological conditions.
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http://dx.doi.org/10.1016/j.isci.2020.100829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993006PMC
February 2020

Regionally specified human pluripotent stem cell-derived astrocytes exhibit different molecular signatures and functional properties.

Development 2019 07 8;146(13). Epub 2019 Jul 8.

Department of Neuroscience, Waisman Center, University of Wisconsin - Madison, Madison, WI 53705, USA

Astrocytes display diverse morphologies in different regions of the central nervous system. Whether astrocyte diversity is attributable to developmental processes and bears functional consequences, especially in humans, is unknown. RNA-seq of human pluripotent stem cell-derived regional astrocytes revealed distinct transcript profiles, suggesting differential functional properties. This was confirmed by differential calcium signaling as well as effects on neurite growth and blood-brain barrier formation. Distinct transcriptional profiles and functional properties of human astrocytes generated from regionally specified neural progenitors under the same conditions strongly implicate the developmental impact on astrocyte diversity. These findings provide a rationale for renewed examination of regional astrocytes and their role in the pathogenesis of psychiatric and neurological disorders.
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http://dx.doi.org/10.1242/dev.170910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633609PMC
July 2019

Generation of seven induced pluripotent stem cell lines from neonates of different ethnic backgrounds.

Stem Cell Res 2019 01 20;34:101365. Epub 2018 Dec 20.

Waisman Center, University of Wisconsin, Madison, WI, USA; Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA. Electronic address:

Seven human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts from three neonatal individuals using non-integrative reprogramming. Most control iPSCs are derived from adults, so these iPSCs meet the need for control iPSCs from young individuals. Donors were from different ethnicities and these lines provide unique genetic profiles. All iPSCs have normal karyotypes, express stem cell markers, and exhibit pluripotency, as assessed by capacity to differentiate into three germ layers. These lines are valuable to study human development, as age-matched controls for disorder-specific iPSCs, and as platforms for gene editing to control for age and ethnicity.
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http://dx.doi.org/10.1016/j.scr.2018.101365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086470PMC
January 2019

Human induced pluripotent stem cell-derived MGE cell grafting after status epilepticus attenuates chronic epilepsy and comorbidities via synaptic integration.

Proc Natl Acad Sci U S A 2019 01 17;116(1):287-296. Epub 2018 Dec 17.

Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX 76502;

Medial ganglionic eminence (MGE)-like interneuron precursors derived from human induced pluripotent stem cells (hiPSCs) are ideal for developing patient-specific cell therapy in temporal lobe epilepsy (TLE). However, their efficacy for alleviating spontaneous recurrent seizures (SRS) or cognitive, memory, and mood impairments has never been tested in models of TLE. Through comprehensive video- electroencephalographic recordings and a battery of behavioral tests in a rat model, we demonstrate that grafting of hiPSC-derived MGE-like interneuron precursors into the hippocampus after status epilepticus (SE) greatly restrained SRS and alleviated cognitive, memory, and mood dysfunction in the chronic phase of TLE. Graft-derived cells survived well, extensively migrated into different subfields of the hippocampus, and differentiated into distinct subclasses of inhibitory interneurons expressing various calcium-binding proteins and neuropeptides. Moreover, grafting of hiPSC-MGE cells after SE mediated several neuroprotective and antiepileptogenic effects in the host hippocampus, as evidenced by reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting in the dentate gyrus (DG). Furthermore, axons from graft-derived interneurons made synapses on the dendrites of host excitatory neurons in the DG and the CA1 subfield of the hippocampus, implying an excellent graft-host synaptic integration. Remarkably, seizure-suppressing effects of grafts were significantly reduced when the activity of graft-derived interneurons was silenced by a designer drug while using donor hiPSC-MGE cells expressing designer receptors exclusively activated by designer drugs (DREADDs). These results implied the direct involvement of graft-derived interneurons in seizure control likely through enhanced inhibitory synaptic transmission. Collectively, the results support a patient-specific MGE cell grafting approach for treating TLE.
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http://dx.doi.org/10.1073/pnas.1814185115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320542PMC
January 2019

Mutations in GFAP Disrupt the Distribution and Function of Organelles in Human Astrocytes.

Cell Rep 2018 10;25(4):947-958.e4

Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Program in Neuroscience & Behavioral Disorders, Duke-NUS Medical School, Singapore, Singapore. Electronic address:

How mutations in glial fibrillary acidic protein (GFAP) cause Alexander disease (AxD) remains elusive. We generated iPSCs from two AxD patients and corrected the GFAP mutations to examine the effects of mutant GFAP on human astrocytes. AxD astrocytes displayed GFAP aggregates, recapitulating the pathological hallmark of AxD. RNA sequencing implicated the endoplasmic reticulum, vesicle regulation, and cellular metabolism. Corroborating this analysis, we observed enlarged and heterogeneous morphology coupled with perinuclear localization of endoplasmic reticulum and lysosomes in AxD astrocytes. Functionally, AxD astrocytes showed impaired extracellular ATP release, which is responsible for attenuated calcium wave propagation. These results reveal that AxD-causing mutations in GFAP disrupt intracellular vesicle regulation and impair astrocyte secretion, resulting in astrocyte dysfunction and AxD pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2018.09.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275075PMC
October 2018

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

Stem Cell Reports 2018 10 27;11(4):998-1008. Epub 2018 Sep 27.

Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; BrainXell, Inc., Madison, WI 53711, USA. Electronic address:

Differentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4-7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.
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http://dx.doi.org/10.1016/j.stemcr.2018.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178885PMC
October 2018

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models.

Mol Cell Proteomics 2018 09 29;17(9):1670-1684. Epub 2018 May 29.

From the ‡Department of Molecular Systems Biology,

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A progenitor cells. Transplantation of human-PSC-derived purified CNTN2 progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.
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http://dx.doi.org/10.1074/mcp.RA118.000809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126395PMC
September 2018

Tissue and cellular rigidity and mechanosensitive signaling activation in Alexander disease.

Nat Commun 2018 05 15;9(1):1899. Epub 2018 May 15.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander disease, and possibly also in other brain disorders characterized by gliosis.
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http://dx.doi.org/10.1038/s41467-018-04269-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954157PMC
May 2018

Modeling Down Syndrome with Patient iPSCs Reveals Cellular and Migration Deficits of GABAergic Neurons.

Stem Cell Reports 2018 04 8;10(4):1251-1266. Epub 2018 Mar 8.

State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China; Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China. Electronic address:

The brain of Down syndrome (DS) patients exhibits fewer interneurons in the cerebral cortex, but its underlying mechanism remains unknown. By morphometric analysis of cortical interneurons generated from DS and euploid induced pluripotent stem cells (iPSCs), we found that DS GABA neurons are smaller and with fewer neuronal processes. The proportion of calretinin over calbindin GABA neurons is reduced, and the neuronal migration capacity is decreased. Such phenotypes were replicated following transplantation of the DS GABAergic progenitors into the mouse medial septum. Gene expression profiling revealed altered cell migratory pathways, and correction of the PAK1 pathway mitigated the cell migration deficit in vitro. These results suggest that impaired migration of DS GABAergic neurons may contribute to the reduced number of interneurons in the cerebral cortex and hippocampus in DS patients.
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http://dx.doi.org/10.1016/j.stemcr.2018.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998838PMC
April 2018

A multi-dimensional characterization of anxiety in monozygotic twin pairs reveals susceptibility loci in humans.

Transl Psychiatry 2017 12 11;7(12):1282. Epub 2017 Dec 11.

Waisman Center, University of Wisconsin, Madison, WI, USA.

The etiology of individual differences in human anxiousness is complex and includes contributions from genetic, epigenetic (i.e., DNA methylation) and environmental factors. Past genomic approaches have been limited in their ability to detect human anxiety-related differences in these factors. To overcome these limitations, we employed both a multi-dimensional characterization method, to select monozygotic twin pairs discordant for anxiety, and whole genome DNA methylation sequencing. This approach revealed 230 anxiety-related differentially methylated loci that were annotated to 183 genes, including several known stress-related genes such as NAV1, IGF2, GNAS, and CRTC1. As an initial validation of these findings, we tested the significance of an overlap of these data with anxiety-related differentially methylated loci that we previously reported from a key neural circuit of anxiety (i.e., the central nucleus of the amygdala) in young monkeys and found a significant overlap (P-value < 0.05) of anxiety-related differentially methylated genes, including GNAS, SYN3, and JAG2. Finally, sequence motif predictions of all the human differentially methylated regions indicated an enrichment of five transcription factor binding motifs, suggesting that DNA methylation may regulate gene expression by mediating transcription factor binding of these transcripts. Together, these data demonstrate environmentally sensitive factors that may underlie the development of human anxiety.
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http://dx.doi.org/10.1038/s41398-017-0047-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802687PMC
December 2017

PSEN1 Mutant iPSC-Derived Model Reveals Severe Astrocyte Pathology in Alzheimer's Disease.

Stem Cell Reports 2017 12 16;9(6):1885-1897. Epub 2017 Nov 16.

A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, 70210 Kuopio, Finland. Electronic address:

Alzheimer's disease (AD) is a common neurodegenerative disorder and the leading cause of cognitive impairment. Due to insufficient understanding of the disease mechanisms, there are no efficient therapies for AD. Most studies have focused on neuronal cells, but astrocytes have also been suggested to contribute to AD pathology. We describe here the generation of functional astrocytes from induced pluripotent stem cells (iPSCs) derived from AD patients with PSEN1 ΔE9 mutation, as well as healthy and gene-corrected isogenic controls. AD astrocytes manifest hallmarks of disease pathology, including increased β-amyloid production, altered cytokine release, and dysregulated Ca homeostasis. Furthermore, due to altered metabolism, AD astrocytes show increased oxidative stress and reduced lactate secretion, as well as compromised neuronal supportive function, as evidenced by altering Ca transients in healthy neurons. Our results reveal an important role for astrocytes in AD pathology and highlight the strength of iPSC-derived models for brain diseases.
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http://dx.doi.org/10.1016/j.stemcr.2017.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785689PMC
December 2017

A neuroprotective astrocyte state is induced by neuronal signal EphB1 but fails in ALS models.

Nat Commun 2017 10 27;8(1):1164. Epub 2017 Oct 27.

John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, E.D. Adrian Building, Forvie Site, Robinson Way, Cambridge, CB2 0PY, UK.

Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1-ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.
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http://dx.doi.org/10.1038/s41467-017-01283-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660125PMC
October 2017

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Adult Common Marmoset Fibroblasts.

Stem Cells Dev 2017 09 24;26(17):1225-1235. Epub 2017 Jul 24.

2 Wisconsin National Primate Research Center, University of Wisconsin-Madison , Madison, Wisconsin.

The common marmoset monkey (Callithrix jacchus; Cj) is an advantageous nonhuman primate species for modeling age-related disorders, including Parkinson's disease, due to their shorter life span compared to macaques. Cj-derived induced pluripotent stem cells (Cj-iPSCs) from somatic cells are needed for in vitro disease modeling and testing regenerative medicine approaches. Here we report the development of a novel Cj-iPSC line derived from adult marmoset fibroblasts. The Cj-iPSCs showed potent pluripotency properties, including the development of mesodermal lineages in tumors after injection to immunocompromised mice, as well as ectoderm and endoderm lineages after in vitro differentiation regimens, demonstrating differentiated derivatives of all three embryonic layers. In addition, expression of key pluripotency genes (ZFP42, PODXL, DNMT3B, C-MYC, LIN28, KLF4, NANOG, SOX2, and OCT4) was observed. We then tested the neural differentiation capacity and gene expression profiles of Cj-iPSCs and a marmoset embryonic stem cell line (Cj-ESC) after dual-SMAD inhibition. Exposure to CHIR99021 and sonic hedgehog (SHH) for 12 and 16 days, respectively, patterned the cells toward a ventralized midbrain dopaminergic phenotype, confirmed by expression of FOXA2, OTX2, EN-1, and tyrosine hydroxylase. These results demonstrate that common marmoset stem cells will be able to serve as a platform for investigating regenerative medicine approaches targeting the dopaminergic system.
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http://dx.doi.org/10.1089/scd.2017.0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576272PMC
September 2017

Sporadic ALS Astrocytes Induce Neuronal Degeneration In Vivo.

Stem Cell Reports 2017 04 30;8(4):843-855. Epub 2017 Mar 30.

Waisman Center, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705, USA; Departments of Neuroscience and Neurology, University of Wisconsin, Madison, WI 53705, USA. Electronic address:

Astrocytes from familial amyotrophic lateral sclerosis (ALS) patients or transgenic mice are toxic specifically to motor neurons (MNs). It is not known if astrocytes from sporadic ALS (sALS) patients cause MN degeneration in vivo and whether the effect is specific to MNs. By transplanting spinal neural progenitors, derived from sALS and healthy induced pluripotent stem cells (iPSCs), into the cervical spinal cord of adult SCID mice for 9 months, we found that differentiated human astrocytes were present in large areas of the spinal cord, replaced endogenous astrocytes, and contacted neurons to a similar extent. Mice with sALS but not non-ALS cells showed reduced non-MNs numbers followed by MNs in the host spinal cord. The surviving MNs showed reduced inputs from inhibitory neurons and exhibited disorganized neurofilaments and aggregated ubiquitin. Correspondingly, mice with sALS but not non-ALS cells showed declined movement deficits. Thus, sALS iPSC-derived astrocytes cause ALS-like degeneration in both MNs and non-MNs.
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http://dx.doi.org/10.1016/j.stemcr.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390239PMC
April 2017

Neural Subtype Specification from Human Pluripotent Stem Cells.

Cell Stem Cell 2016 11;19(5):573-586

Waisman Center, University of Wisconsin, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA. Electronic address:

Human pluripotent stem cells (hPSCs) provide a model to study early neural development, model pathological processes, and develop therapeutics. The generation of functionally specialized neural subtypes from hPSCs relies on fundamental developmental principles learned from animal studies. Manipulation of these principles enables production of highly enriched neural types with functional attributes that resemble those in the brain. Further development to promote faster maturation or aging as well as circuit integration will help realize the potential of hPSC-derived neural cells in disease modeling and cell therapy.
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http://dx.doi.org/10.1016/j.stem.2016.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127287PMC
November 2016

Real-Time Intraoperative MRI Intracerebral Delivery of Induced Pluripotent Stem Cell-Derived Neurons.

Cell Transplant 2017 04 14;26(4):613-624. Epub 2016 Sep 14.

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson's disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.
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http://dx.doi.org/10.3727/096368916X692979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539906PMC
April 2017

Time-Course Gene Expression Profiling Reveals a Novel Role of Non-Canonical WNT Signaling During Neural Induction.

Sci Rep 2016 09 7;6:32600. Epub 2016 Sep 7.

Waisman Center, University of Wisconsin, Madison, WI 53705, USA.

The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-β and canonical WNT/β-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca(2+) signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca(2+) pathway, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca(2+)/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation.
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http://dx.doi.org/10.1038/srep32600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013468PMC
September 2016

Chemical Control of Grafted Human PSC-Derived Neurons in a Mouse Model of Parkinson's Disease.

Cell Stem Cell 2016 06 28;18(6):817-826. Epub 2016 Apr 28.

Waisman Center, University of Wisconsin-Madison, Madison, WI 53705, USA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA; Department of Neurology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA. Electronic address:

Transplantation of human pluripotent stem cell (hPSC)-derived neurons is a promising avenue for treating disorders including Parkinson's disease (PD). Precise control over engrafted cell activity is highly desired, as cells do not always integrate properly into host circuitry and can cause suboptimal graft function or undesired outcomes. Here, we show tunable rescue of motor function in a mouse model of PD, following transplantation of human midbrain dopaminergic (mDA) neurons differentiated from hPSCs engineered to express DREADDs (designer receptors exclusively activated by designer drug). Administering clozapine-N-oxide (CNO) enabled precise DREADD-dependent stimulation or inhibition of engrafted neurons, revealing D1 receptor-dependent regulation of host neuronal circuitry by engrafted cells. Transplanted cells rescued motor defects, which could be reversed or enhanced by CNO-based control of graft function, and activating engrafted cells drives behavioral changes in transplanted mice. These results highlight the ability to exogenously and noninvasively control and refine therapeutic outcomes following cell transplantation.
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http://dx.doi.org/10.1016/j.stem.2016.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892985PMC
June 2016

Engineering human cells and tissues through pluripotent stem cells.

Curr Opin Biotechnol 2016 08 12;40:133-138. Epub 2016 Apr 12.

Waisman Center, University of Wisconsin, Madison, WI 53705, United States; Department of Neuroscience, University of Wisconsin, Madison, WI 53705, United States; Department of Neurology, University of Wisconsin, Madison, WI 53705, United States. Electronic address:

The utility of human pluripotent stem cells (hPSCs) depends on their ability to produce functional cells and tissues of the body. Two strategies have been developed: directed differentiation of enriched populations of cells that match a regional and functional profile and spontaneous generation of three-dimensional organoids that resemble tissues in the body. Genomic editing of hPSCs and their differentiated cells broadens the use of the hPSC paradigm in studying human cellular function and disease as well as developing therapeutics.
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http://dx.doi.org/10.1016/j.copbio.2016.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975619PMC
August 2016

Modeling ALS with motor neurons derived from human induced pluripotent stem cells.

Nat Neurosci 2016 Apr;19(4):542-53

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA.

Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.
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http://dx.doi.org/10.1038/nn.4273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015775PMC
April 2016

Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration.

Nat Med 2016 05 28;22(5):479-87. Epub 2016 Mar 28.

Department of Neurosciences, University of California, San Diego, La Jolla, California, USA.

The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In murine models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of SCI. Corticospinal regeneration requires grafts to be driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, a process that is potentially related to the attenuation of the glial scar. Rat corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord 'replacement' with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of SCI research and offering new possibilities for clinical translation.
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http://dx.doi.org/10.1038/nm.4066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860037PMC
May 2016

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

Electrophoresis 2016 Apr 24;37(7-8):1027-30. Epub 2016 Feb 24.

School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus.
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http://dx.doi.org/10.1002/elps.201500496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314833PMC
April 2016

Generation of serotonin neurons from human pluripotent stem cells.

Nat Biotechnol 2016 Jan 14;34(1):89-94. Epub 2015 Dec 14.

Waisman Center, University of Wisconsin, Madison, Wisconsin, USA.

Serotonin neurons located in the raphe nucleus of the hindbrain have crucial roles in regulating brain functions and have been implicated in various psychiatric disorders. Yet functional human serotonin neurons are not available for in vitro studies. Through manipulation of the WNT pathway, we demonstrate efficient differentiation of human pluripotent stem cells (hPSCs) to cells resembling central serotonin neurons, primarily those located in the rhombomeric segments 2-3 of the rostral raphe, which participate in high-order brain functions. The serotonin neurons express a series of molecules essential for serotonergic development, including tryptophan hydroxylase 2, exhibit typical electrophysiological properties and release serotonin in an activity-dependent manner. When treated with the FDA-approved drugs tramadol and escitalopram oxalate, they release or uptake serotonin in a dose- and time-dependent manner, suggesting the utility of these cells for the evaluation of drug candidates.
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http://dx.doi.org/10.1038/nbt.3435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711820PMC
January 2016

Spinal muscular atrophy patient-derived motor neurons exhibit hyperexcitability.

Sci Rep 2015 Jul 20;5:12189. Epub 2015 Jul 20.

1] Waisman center, University of Wisconsin, Madison, WI, 53705, USA [2] Department of Neuroscience and Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705, USA.

Spinal muscular atrophy (SMA) presents severe muscle weakness with limited motor neuron (MN) loss at an early stage, suggesting potential functional alterations in MNs that contribute to SMA symptom presentation. Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs. We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs. Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.
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http://dx.doi.org/10.1038/srep12189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507262PMC
July 2015
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