scholarly journals In vivo modeling of human neuron dynamics and Down syndrome

Science ◽  
2018 ◽  
Vol 362 (6416) ◽  
pp. eaau1810 ◽  
Author(s):  
Raquel Real ◽  
Manuel Peter ◽  
Antonio Trabalza ◽  
Shabana Khan ◽  
Mark A. Smith ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Cortical Neurons ◽  
Synapse Formation ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Human Stem Cells ◽  
Population Activity ◽  
Human Neurons ◽  
Longitudinal Imaging

Harnessing the potential of human stem cells for modeling the physiology and diseases of cortical circuitry requires monitoring cellular dynamics in vivo. We show that human induced pluripotent stem cell (iPSC)–derived cortical neurons transplanted into the adult mouse cortex consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories with complex cytoarchitecture. Longitudinal imaging of >4000 grafted developing human neurons revealed that neuronal arbors refined via branch-specific retraction; human synaptic networks substantially restructured over 4 months, with balanced rates of synapse formation and elimination; and oscillatory population activity mirrored the patterns of fetal neural networks. Lastly, we found increased synaptic stability and reduced oscillations in transplants from two individuals with Down syndrome, demonstrating the potential of in vivo imaging in human tissue grafts for patient-specific modeling of cortical development, physiology, and pathogenesis.

Abstract 518: In Vitro and in vivo Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Cynthia St. Hilaire ◽  
Hui Jin ◽  
Yuting Huang ◽  
Dan Yang ◽  
Alejandra Negro ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Dermal Fibroblasts ◽  
In Vivo Studies ◽  
Arterial Calcification ◽  
Patient Specific ◽  
And Control

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

scholarly journals Presynaptic dysfunction in CASK-related neurodevelopmental disorders

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Becker ◽  
Francesca Mastropasqua ◽  
Jan Philipp Reising ◽  
Simon Maier ◽  
Mai-Lan Ho ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Splice Site Mutation ◽  
Autism Spectrum ◽  
Resonance Spectroscopy ◽  
Limited Information ◽  
Site Mutation ◽  
Mutation Carriers ◽  
Human Neurons

Abstract CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory–inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.

Assessment of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Contractility Using Micropost Arrays

2013 ◽  
Author(s):  
Marita L. Rodriguez ◽  
Charles E. Murry ◽  
Nathan J. Sniadecki
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Patient Specific ◽  
Human Stem Cells ◽  
Cell Therapies ◽  
Cardiomyocyte Contractility ◽  
Induced Pluripotent ◽  
Contractile Forces

Cardiovascular stem cell therapies have shown increasing promise as a potential therapeutic means for reversing the effects of a myocardial infarction [1]. Out of the currently available sources of human stem cells, human induced pluripotent stem cells (hiPSCs) are very promising in that: the number of cell lines that can be induced to the pluripotent state is extremely vast, they serve as a potential source for patient-specific cardiomyocytes, and their use is non-controversial. However, before they can be used feasibly in a clinical setting, the functional engraftment of these cells into the host tissue must be improved [2]. It is hypothesized that the structural and functional maturity of the stem-cell derived cardiomyocytes prior to implantation, may significantly affect the ability of these cells to engraft with resident heart tissue [3]. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. However, assessing the contractile properties of single iPS-CMs is a difficult task. iPS-CMs generally have relatively unorganized cytoskeletons, with stress fibers in multiple directions. This trait renders one or two-point force assays ineffectual in determining total cell forces. Furthermore, iPS-CMs don’t spread well on tissue culture surfaces, which make two-dimensional force measurements almost impossible.

scholarly journals Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived from human iPSCs

2021 ◽  
Author(s):  
Zhefu Que ◽  
Maria I. Olivero-Acosta ◽  
Jingliang Zhang ◽  
Muriel Eaton ◽  
William C. Skarnes ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Cortical Neurons ◽  
Sodium Current ◽  
Electrode Array ◽  
Induced Pluripotent Stem Cell ◽  
Epileptic Seizures ◽  
Action Potential Firing ◽  
Human Neurons ◽  
Human Ipscs

AbstractWith the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

scholarly journals Fezf2 transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

2020 ◽  
Author(s):  
Cameron Sadegh ◽  
Wataru Ebina ◽  
Anthony C. Arvanites ◽  
Lance S. Davidow ◽  
Lee L. Rubin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cortical Neurons ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Sirtuin 1 ◽  
Projection Neurons ◽  
Specific Expression ◽  
Specific Differentiation

AbstractDuring late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

scholarly journals An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

2020 ◽  
Author(s):  
◽  
Loren Ornelas ◽  
Emilda Gomez ◽  
Lindsay Panther ◽  
Aaron Frank ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Rna Splicing ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Data Independent Acquisition ◽  
Induced Pluripotent ◽  
Als Patients ◽  
And Control

SummaryNeurodegenerative diseases present a challenge for systems biology, due to the lack of reliable animal models and the difficulties in obtaining samples from patients at early stages of disease, when interventions might be most effective. Studying induced pluripotent stem cell (iPSC)-derived neurons could overcome these challenges and dramatically accelerate and broaden therapeutic strategies. Here we undertook a network-based multi-omic characterization of iPSC-derived motor neurons from ALS patients carrying genetically dominant hexanucleotide expansions in C9orf72 to gain a deeper understanding of the relationship between DNA, RNA, epigenetics and protein in the same pool of tissue. ALS motor neurons showed the expected C9orf72-related alterations to specific nucleoporins and production of dipeptide repeats. RNA-seq, ATAC-seq and data-independent acquisition mass-spectrometry (DIA-MS) proteomics were then performed on the same motor neuron cultures. Using integrative computational methods that combined all of the omics, we discovered a number of novel dysregulated pathways including biological adhesion and extracellular matrix organization and disruption in other expected pathways such as RNA splicing and nuclear transport. We tested the relevance of these pathways in vivo in a C9orf72 Drosophila model, analyzing the data to determine which pathways were causing disease phenotypes and which were compensatory. We also confirmed that some pathways are altered in late-stage neurodegeneration by analyzing human postmortem C9 cervical spine data. To validate that these key pathways were integral to the C9 signature, we prepared a separate set of C9orf72 and control motor neuron cultures using a different differentiation protocol and applied the same methods. As expected, there were major overall differences between the differentiation protocols, especially at the level of in individual omics data. However, a number of the core dysregulated pathways remained significant using the integrated multiomic analysis. This new method of analyzing patient specific neural cultures allows the generation of disease-related hypotheses with a small number of patient lines which can be tested in larger cohorts of patients.

scholarly journals MiR-33a is a therapeutic target in SPG4-related hereditary spastic paraplegia human neurons

2019 ◽  
Vol 133 (4) ◽  
pp. 583-595 ◽  
Author(s):  
Fumiko Nakazeki ◽  
Itaru Tsuge ◽  
Takahiro Horie ◽  
Keiko Imamura ◽  
Kayoko Tsukita ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Cortical Neurons ◽  
Hereditary Spastic Paraplegia ◽  
Regulatory Element ◽  
Induced Pluripotent Stem Cell ◽  
Cholesterol Homeostasis ◽  
Locked Nucleic Acid ◽  
Spastic Paraplegia ◽  
Potential Therapeutic Target ◽  
Human Neurons

Abstract Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3′-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Olivia T Ly ◽  
Grace Brown ◽  
Hanna Chen ◽  
Liang Hong ◽  
Xinge Wang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Combinatorial Approach ◽  
Atrial Cardiomyocytes ◽  
Myocardial Substrate ◽  
Induced Pluripotent

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

Abstract 14013: Direct Comparison of Disease-Specific versus TALEN-Corrected iPSC-Derived Cardiomyocytes from Dilated Cardiomyopathy Patients

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Antje Ebert ◽  
Johannes Riegler ◽  
Ioannis Karakikes ◽  
Vittavat Termglinchan ◽  
Mohammed Mameen ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Heart Function ◽  
Myocardial Infarct ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Transcription Activator ◽  
Significant Difference ◽  
Healthy Control

Introduction: Recent advances in regenerative medicine for cardiovascular disease (CVD) therapy focus on delivery of autologous induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to recover heart function without immunosuppression. Here, we perform a direct comparison of iPSC-CMs from a healthy individual, a DCM patient, and following genome-correction with transcription activator-like effector nucleases (TALENs) both in vitro and in vivo. Methods & Results: We established patient-specific iPSC-CMs that recapitulate DCM disease-phenotypes in-vitro, including disrupted sarcomeres and impaired Ca2+ handling (p<0.05). We corrected the patient′s DCM-specific mutation R173W in the TNNT2 locus via TALEN-mediated footprint-free genome editing. Isogenic TALEN-corrected iPSC-CMs recovered functional properties comparable to healthy control. Ca2+ transient analysis revealed no significant difference between TALEN-corrected iPSC-CMs and healthy control regarding amplitude (ΔF/F0 4.71±0.55), decay (277.6 ±28.2 ms), and time to peak (240.5±22.5 ms). Confocal microscopy of TALEN-corrected iPSC-CMs confirmed sarcomeric structures as in healthy control (Fig 1). We transplanted (i) 1x10*7 DCM iPSC-CMs, (ii) 1x10*7 TALEN-corrected iPSC-CMs, and (iii) 1x10*7 healthy control iPSC-CMs into a subacute myocardial infarct (MI) rat model (n=15/group). Cell engraftment into the host myocardium was confirmed by immunohistochemistry. Comprehensive functional analysis via magnetic resonance imaging (MRI), echocardiography is underway. Conclusions: We generated TALEN-corrected iPSC-CMs from a DCM patient, which recover in vitro functional parameters of healthy control iPSC-CMs. We transplanted for the first time patient-specific and TALEN-corrected iPSC-CMs into a rodent MI model. This approach may represent a viable option for mechanistic analysis and regenerative medicine in CVD patients. Figure 1:

scholarly journals Human iPSC-derived trigeminal neurons lack constitutive TLR3-dependent immunity that protects cortical neurons from HSV-1 infection

2018 ◽  
Vol 115 (37) ◽  
pp. E8775-E8782 ◽  
Author(s):  
Bastian Zimmer ◽  
Osefame Ewaleifoh ◽  
Oliver Harschnitz ◽  
Yoon-Seung Lee ◽  
Camille Peneau ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Poly I:C ◽  
Inborn Errors ◽  
Constitutive Resistance ◽  
Human Ipscs ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/β– and -λ–inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti–HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-β but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/β receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.

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