Journal articles on the topic 'IPSC-derived MN'
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1
Castellanos-Montiel, María José, Mathilde Chaineau, Anna Kristyna Franco-Flores, Ghazal Haghi, Dulce Carrillo-Valenzuela, Wolfgang E. Reintsch, Carol X. Q. Chen, and Thomas M. Durcan. "An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids." Cells 12, no. 4 (February 8, 2023): 545. http://dx.doi.org/10.3390/cells12040545.
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A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time.
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Zhang, Yiti, Baitao Zeng, Ao Gu, Qinyu Kang, Mingri Zhao, Guangnan Peng, Miaojin Zhou, et al. "Damaged DNA Is an Early Event of Neurodegeneration in Induced Pluripotent Stem Cell-Derived Motoneurons with UBQLN2P497H Mutation." International Journal of Molecular Sciences 23, no. 19 (September 26, 2022): 11333. http://dx.doi.org/10.3390/ijms231911333.
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Ubiquilin-2 (UBQLN2) mutations lead to familial amyotrophic lateral sclerosis (FALS)/and frontotemporal dementia (FTLD) through unknown mechanisms. The combination of iPSC technology and CRISPR-mediated genome editing technology can generate an iPSC-derived motor neuron (iPSC-MN) model with disease-relevant mutations, which results in increased opportunities for disease mechanism research and drug screening. In this study, we introduced a UBQLN2-P497H mutation into a healthy control iPSC line using CRISPR/Cas9, and differentiated into MNs to study the pathology of UBQLN2-related ALS. Our in vitro MN model faithfully recapitulated specific aspects of the disease, including MN apoptosis. Under sodium arsenite (SA) treatment, we found differences in the number and the size of UBQLN2+ inclusions in UBQLN2P497H MNs and wild-type (WT) MNs. We also observed cytoplasmic TAR DNA-binding protein (TARDBP, also known as TDP-43) aggregates in UBQLN2P497H MNs, but not in WT MNs, as well as the recruitment of TDP-43 into stress granules (SGs) upon SA treatment. We noted that UBQLN2-P497H mutation induced MNs DNA damage, which is an early event in UBQLN2-ALS. Additionally, DNA damage led to an increase in compensation for FUS, whereas UBQLN2-P497H mutation impaired this function. Therefore, FUS may be involved in DNA damage repair signaling.
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Gori, Jennifer L., Devikha Chandrasekaran, John P. Kowalski, Jennifer E. Adair, Brian C. Beard, Sunita L. D'Souza, and Hans-Peter Kiem. "Efficient generation, purification, and expansion of CD34+ hematopoietic progenitor cells from nonhuman primate–induced pluripotent stem cells." Blood 120, no. 13 (September 27, 2012): e35-e44. http://dx.doi.org/10.1182/blood-2012-05-433797.
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AbstractInduced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34+CD38−Thy1+CD45RA−CD49f+ cell yield by 6-fold. CD34+CD38−Thy1+CD45RA−CD49f+ cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34high cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34+ cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34high cell therapies in a clinically relevant nonhuman primate model.
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Thiry, Louise, Jean-Pierre Clément, Rainer Haag, Timothy E. Kennedy, and Stefano Stifani. "Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate." ASN Neuro 14 (January 2022): 175909142110733. http://dx.doi.org/10.1177/17590914211073381.
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Human induced pluripotent stem cells (hiPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on hiPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using hiPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current hiPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of hiPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve hiPSC-based studies of human MN biology and disease. We describe the use of a new coating substrate providing improved conditions for long-term cultures of human iPSC-derived motor neurons, thus allowing evaluation of cell viability, molecular identity, spontaneous network electrophysiological activity, and single-cell RNA sequencing of mature motor neurons.
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Johns, Alexandra E., and Nicholas J. Maragakis. "Exploring Motor Neuron Diseases Using iPSC Platforms." Stem Cells 40, no. 1 (January 1, 2022): 2–13. http://dx.doi.org/10.1093/stmcls/sxab006.
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Abstract The degeneration of motor neurons is a pathological hallmark of motor neuron diseases (MNDs), but emerging evidence suggests that neuronal vulnerability extends well beyond this cell subtype. The ability to assess motor function in the clinic is limited to physical examination, electrophysiological measures, and tissue-based or neuroimaging techniques which lack the resolution to accurately assess neuronal dysfunction as the disease progresses. Spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), hereditary spastic paraplegia (HSP), and amyotrophic lateral sclerosis (ALS) are all MNDs with devastating clinical outcomes that contribute significantly to disease burden as patients are no longer able to carry out normal activities of daily living. The critical need to accurately assess the cause and progression of motor neuron dysfunction, especially in the early stages of those diseases, has motivated the use of human iPSC-derived motor neurons (hiPSC-MN) to study the neurobiological mechanisms underlying disease pathogenesis and to generate platforms for therapeutic discovery and testing. As our understanding of MNDs has grown, so too has our need to develop more complex in vitro models which include hiPSC-MN co-cultured with relevant non-neuronal cells in 2D as well as in 3D organoid and spheroid systems. These more complex hiPSC-derived culture systems have led to the implementation of new technologies, including microfluidics, multielectrode array, and machine learning which offer novel insights into the functional correlates of these emerging model systems.
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Akter, Masuma, and Baojin Ding. "Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons." Cells 11, no. 23 (November 27, 2022): 3796. http://dx.doi.org/10.3390/cells11233796.
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Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions.
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Watts, Michelle E., Chen Wu, and Lee L. Rubin. "Suppression of MAP4K4 Signaling Ameliorates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis-Molecular Studies Toward New Therapeutics." Journal of Experimental Neuroscience 13 (January 2019): 117906951986279. http://dx.doi.org/10.1177/1179069519862798.
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Amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease of adults, is characterized by the degeneration of upper MNs in the motor cortex and lower MNs in the brain stem and spinal cord. Our recent work suggests that a MAP kinase family member, MAP4K4 (mitogen-activated protein kinase kinase kinase kinase 4), regulates MN degeneration in ALS. Activation of MAP4K4 occurs prior to MN death and inhibition of MAP4K4 improves neurite integrity and neuronal viability in a cell autonomous manner. The mechanism through which MAP4K4 reduction specifically modulates MN viability can be attributed to the attenuation of the c-Jun apoptotic pathway, as well as to the activation of FoxO1-mediated autophagy that reduces the accumulation of protein aggregates. We additionally show the feasibility of MAP4K4 as a drug target using a MAP4K4-specific inhibitor, which improves the survival of both primary and induced pluripotent stem cell (iPSC)-derived MNs. Our studies are thus far the first to highlight a MAP4K4-initiated signaling cascade that contributes to MN degeneration in ALS, providing a new mechanism underlying MN death in disease and a druggable target for new therapeutics. We propose exciting future directions and unexplored avenues based upon this work.
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Bräuer, Stefan, René Günther, Jared Sterneckert, Hannes Glaß, and Andreas Hermann. "Human Spinal Motor Neurons Are Particularly Vulnerable to Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients." International Journal of Molecular Sciences 21, no. 10 (May 18, 2020): 3564. http://dx.doi.org/10.3390/ijms21103564.
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Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its pathophysiological cascade is still enigmatic. More than 90% of ALS patients suffer from sporadic ALS, which makes it specifically demanding to generate appropriate model systems. One interesting aspect considering the seeding, spreading and further disease development of ALS is the cerebrospinal fluid (CSF). We therefore asked whether CSF from sporadic ALS patients is capable of causing disease typical changes in human patient-derived spinal MN cultures and thus could represent a novel model system for sporadic ALS. By using induced pluripotent stem cell (iPSC)-derived MNs from healthy controls and monogenetic forms of ALS we could demonstrate a harmful effect of ALS-CSF on healthy donor-derived human MNs. Golgi fragmentation—a typical finding in lower organism models and human postmortem tissue—was induced solely by addition of ALS-CSF, but not control-CSF. No other neurodegenerative hallmarks—including pathological protein aggregation—were found, underpinning Golgi fragmentation as early event in the neurodegenerative cascade. Of note, these changes occurred predominantly in MNs, the cell type primarily affected in ALS. We thus present a novel way to model early features of sporadic ALS.
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Budding, Kevin, Lill Eva Johansen, Inge Van de Walle, Kim Dijkxhoorn, Elisabeth de Zeeuw, Lauri M. Bloemenkamp, Jeroen W. Bos, et al. "Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy." Neurology - Neuroimmunology Neuroinflammation 9, no. 1 (November 10, 2021): e1107. http://dx.doi.org/10.1212/nxi.0000000000001107.
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Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.
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Jin, Mengmeng, Katja Akgün, Tjalf Ziemssen, Markus Kipp, Rene Günther, and Andreas Hermann. "Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs." International Journal of Molecular Sciences 22, no. 15 (July 27, 2021): 8042. http://dx.doi.org/10.3390/ijms22158042.
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Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.
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Telliam, Gladys, Christophe Desterke, Olivier Féraud, Frank Griscelli, Noufissa Oudrhiri, Micheline Fontaine Arnoux, Radhia Najar, Herve Acloque, Annelise Bennaceur-Griscelli, and Ali G. Turhan. "Blast Crisis in a Dish: Generation of a Blast Crisis Model in Chronic Myeloid Leukemia (CML) Using Patient-Specific Induced Pluripotent Stem Cells ( iPSC)." Blood 128, no. 22 (December 2, 2016): 933. http://dx.doi.org/10.1182/blood.v128.22.933.933.
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Abstract Despite the major progress obtained in prognosis with the use of tyrosine kinase inhibitors (TKI), the great majority of patients with CML remain on long-term therapy and progression occurs in patients with either primary or secondary resistance. The mechanisms of progression towards accelerated phase (AP) and blast crisis (BC) have been studied so far only in primary patient samples in BC. Currently, there is no in vitro model to study sequentially the molecular events leading from CP to BC as only some primary sequential samples are amenable to analysis. Using induced Pluripotent Stem Cell (iPSC) technology, it is now possible to reprogram the primary leukemic cells to pluripotency and generate a major source of stem cells. To determine the feasibility of studying progression of CML towards AP and BC, we have used a patient-specific iPSC line that we have generated from the primary leukemic cells of a patient who later showed a TKI-resistance requiring an allogeneic stem cell transplant. These iPSC expressed BCR-ABL, exhibited all pluripotency markers and after injection in NSG mice, generated teratoma with differentiation into three germ layers. In hematopoietic differentiation assays using day 19-embryoid bodies (EB), increased numbers of hematopoietic progenitors were found as compared to control iPSC (5-fold increase n= 3). We have then treated leukemic iPSC with the mutagenic agent N-ethyl-N-nitrosourea (ENU) during regular medium changes. After 61 days in ENU cultures, day-19 derived embryoid bodies generated hematopoietic cells (>90% CD45+, CD43+) which proliferated in liquid cultures with myeloid and some blast cell morphology. Cytogenetic analyses of iPSC revealed chromosomal abnormalities such as loss of Y and loss of der q9+, both alterations known to occur in CML during progression. They exhibited increased numbers of micronuclei (MN) as compared to leukemic iPSC without ENU (X 3 Fold increase) suggesting acquisition of a progressive chromosomal instability. CGH array analyses were performed using ENU-treated iPSC-derived hematopoietic cells in two different timepoints as compared to leukemic iPSC cultured without ENU. Genomic aberrations were analyzed by Agilent Cytogenomics software with Mosaicism workflow on HG19 genome. 249 gene loci alterations were detected after polymorphism filtration on European population. These analyses showed DNA losses and DNA gains in genes implicated in mesoderm development and hematopoietic lineage as well as genes implicated in DNA damage response. Several loci of transcription factors were found to be involved such as IKZF1 described in imatinib-refractory chronic myeloid leukemia (Bolton-Gillespie et al. 2013). The aberrations included SIRT1and BLM which is implicated in DNA repair. Several cancer genes were found to be involved, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Remarkably, transcriptome geodataset GSE4170 (Radich et al. 2006) allowed us to associate 125 of 249 of the aberrations that we detected in CML iPSC, with the CML progression genes already described during progression from chronic and AP to BC (p-value =9.43E-32, after ANOVA with 1000 permutations). 38 most predictive aberrations allowed perfect reclassification of BC and chronic phase samples by unsupervised classification. Among these candidates, eleven of them have been described in CML physiopathology and connected to TKI resistance and genomic instability. Majority of them ( 7/11) are connected to chronic phase (FAS, ACTB, TRIM21, ANPEP, MLK1, CSF2RA, and MAGEC2) whereas a minority of them (4/11) are connected to BC (ACP1, SH3YL1, FHL1, IL3RA). Interestingly, these experiments also allowed us to discover the connection of a new multidrug resistance molecule associated to BC and having the ability to modify interferon pathway connected to the TKI sensitivity. Thus, genomic instability pattern that we have generated using a single leukemic iPSC allowed duplication of genomic abnormalities described in CML progression and allowed identification of some novel genes. Overall, these results demonstrated that we have generated for the first time to our knowledge, an in vitro BC model, reproducing genomic events described in patients with BC. This "blast crisis in a dish" tool using patient-derived iPSC will be of major interest to study CML progression and eventually to test novel therapies. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Sepehrimanesh, Masood, and Baojin Ding. "Generation and optimization of highly pure motor neurons from human induced pluripotent stem cells via lentiviral delivery of transcription factors." American Journal of Physiology-Cell Physiology 319, no. 4 (October 1, 2020): C771—C780. http://dx.doi.org/10.1152/ajpcell.00279.2020.
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Generation of neurons from human induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain samples and greatly facilitates the progress of research in neurological diseases. However, it is still a challenge to generate a particular neuronal subtype with high purity and yield for determining the pathogenesis of diseased neurons using biochemical approaches. Motor neurons (MNs) are a specialized neuronal subtype responsible for governing both autonomic and volitional movement. Dysfunctions in MNs are implicated in a variety of movement diseases, such as amyotrophic lateral sclerosis (ALS). In this study, we generated functional MNs from human iPSCs via lentiviral delivery of transcription factors. Moreover, we optimized induction conditions by using different combinations of transcription factors and found that a single lentiviral vector expressing three factors [neurogenin-2 (NGN2), insulin gene enhancer 1 (ISL1), and LIM/homeobox 3 (LHX3)] is necessary and sufficient to induce iPSC-derived MNs (iPSC-MNs). These MNs robustly expressed general neuron markers [microtubule-associated protein 2 (MAP2), neurofilament protein (SMI-32), and tubulin β-3 class III (TUBB3)] and MN-specific markers [HB9 and choline acetyltransferase (ChAT)] and showed electrical maturation and firing of action potentials within 3 wk. This approach significantly improved the neuronal survival, yield, and purity, making it feasible to obtain abundant materials for biochemical studies in modeling movement diseases.
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Osaki, Tatsuya, Sebastien G. M. Uzel, and Roger D. Kamm. "Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons." Science Advances 4, no. 10 (October 2018): eaat5847. http://dx.doi.org/10.1126/sciadv.aat5847.
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Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease involving loss of motor neurons (MNs) and muscle atrophy, still has no effective treatment, despite much research effort. To provide a platform for testing drug candidates and investigating the pathogenesis of ALS, we developed an ALS-on-a-chip technology (i.e., an ALS motor unit) using three-dimensional skeletal muscle bundles along with induced pluripotent stem cell (iPSC)–derived and light-sensitive channelrhodopsin-2–induced MN spheroids from a patient with sporadic ALS. Each tissue was cultured in a different compartment of a microfluidic device. Axon outgrowth formed neuromuscular junctions on the muscle fiber bundles. Light was used to activate muscle contraction, which was measured on the basis of pillar deflections. Compared to a non-ALS motor unit, the ALS motor unit generated fewer muscle contractions, there was MN degradation, and apoptosis increased in the muscle. Furthermore, the muscle contractions were recovered by single treatments and cotreatment with rapamycin (a mechanistic target of rapamycin inhibitor) and bosutinib (an Src/c-Abl inhibitor). This recovery was associated with up-regulation of autophagy and degradation of TAR DNA binding protein–43 in the MNs. Moreover, administering the drugs via an endothelial cell barrier decreased the expression of P-glycoprotein (an efflux pump that transports bosutinib) in the endothelial cells, indicating that rapamycin and bosutinib cotreatment has considerable potential for ALS treatment. This ALS-on-a-chip and optogenetics technology could help to elucidate the pathogenesis of ALS and to screen for drug candidates.
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Gori, Jennifer L., Jason M. Butler, Devikha Chandrasekaran, Brian C. Beard, Daniel J. Nolan, Michael Ginsberg, Jennifer E. Adair, Shahin Rafii, and Hans-Peter Kiem. "In Vivo Selection and Long-Term Engraftment Of Hematopoietic Stem Cells Generated Via Vascular Niche Induction Of Nonhuman Primate Induced Pluripotent Stem Cells." Blood 122, no. 21 (November 15, 2013): 466. http://dx.doi.org/10.1182/blood.v122.21.466.466.
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Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P<0.05). On average, 15% CD34+ and 37% CD45+ cells were detected in the bone marrow of mice transplanted with E4ORF1+EC-expanded P140K-MniPSC HSCs, which is significantly higher than levels detected in the other cohorts (Table 1). CD45+ cells in the marrow were predominantly myeloid but lymphoid subsets were also present (10-25% CD3+ cells). Remarkably, the level of gene marking in CFCs and number of gene marked CFCs from mouse bone marrow was substantially higher for mice transplanted with E4ORF1+EC expanded compared to cytokine expanded P140K-MniPSC-HSCs (Table 1). Finally, to confirm engraftment of authentic HSCs, secondary transplants were established. Although engraftment was achieved in all secondary transplanted cohorts, the level of nonhuman primate cells detected was significantly higher in animals transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs. Significantly more lymphocytes (CD45+CD3+ and CD45+CD56+) and monocytes (CD45+CD14+) were detected in the blood of these secondary transplant recipients. These findings confirm generation of bona fide HSCs derived from nonhuman primate iPSCs and demonstrate that O6BG/BCNU chemotherapy supports in vivo selection of P140K-MniPSC-HSCs generated by co-culture with the E4ORF1+EC vascular platform. Our studies mark a significant advance toward clinical translation of PSC-based blood therapeutics and the development of a nonhuman primate preclinical model. Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.
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Ting, Hsiao-Chien, Hui-I. Yang, Horng-Jyh Harn, Ing-Ming Chiu, Hong-Lin Su, Xiang Li, Mei-Fang Chen, et al. "Coactivation of GSK3β and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons." Cells 10, no. 10 (October 16, 2021): 2773. http://dx.doi.org/10.3390/cells10102773.
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Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients’ MNs, SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1G85R and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1G93A ALS mice. Moreover, we found that coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3β activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3β and IGF-1 activation) that may serve as potential targets for ALS therapy.
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Burley, Sarah, Dayne A. Beccano-Kelly, Kevin Talbot, Oscar Cordero Llana, and Richard Wade-Martins. "Hyperexcitability in young iPSC-derived C9ORF72 mutant motor neurons is associated with increased intracellular calcium release." Scientific Reports 12, no. 1 (May 5, 2022). http://dx.doi.org/10.1038/s41598-022-09751-3.
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AbstractA large hexanucleotide repeat expansion in the C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca2+) from internal stores, but with no changes to NaV and KV currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca2+ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.
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Dodge, James C., Jinlong Yu, S. Pablo Sardi, and Lamya S. Shihabuddin. "Sterol auto-oxidation adversely affects human motor neuron viability and is a neuropathological feature of amyotrophic lateral sclerosis." Scientific Reports 11, no. 1 (January 12, 2021). http://dx.doi.org/10.1038/s41598-020-80378-y.
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AbstractAberrant cholesterol homeostasis is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal neuromuscular disease that is due to motor neuron (MN) death. Cellular toxicity from excess cholesterol is averted when it is enzymatically oxidized to oxysterols and bile acids (BAs) to promote its removal. In contrast, the auto oxidation of excess cholesterol is often detrimental to cellular survival. Although oxidized metabolites of cholesterol are altered in the blood and CSF of ALS patients, it is unknown if increased cholesterol oxidation occurs in the SC during ALS, and if exposure to oxidized cholesterol metabolites affects human MN viability. Here, we show that in the SOD1G93A mouse model of ALS that several oxysterols, BAs and auto oxidized sterols are increased in the lumbar SC, plasma, and feces during disease. Similar changes in cholesterol oxidation were found in the cervical SC of sporadic ALS patients. Notably, auto-oxidized sterols, but not oxysterols and BAs, were toxic to iPSC derived human MNs. Thus, increased cholesterol oxidation is a manifestation of ALS and non-regulated sterol oxidation likely contributes to MN death. Developing therapeutic approaches to restore cholesterol homeostasis in the SC may lead to a treatment for ALS.
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Solomon, Emilia, Katie Davis-Anderson, Blake Hovde, Sofiya Micheva-Viteva, Jennifer Foster Harris, Scott Twary, and Rashi Iyer. "Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation." BMC Molecular and Cell Biology 22, no. 1 (February 18, 2021). http://dx.doi.org/10.1186/s12860-021-00343-z.
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Abstract Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.
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Vandoorne, Tijs, Koen Veys, Wenting Guo, Adria Sicart, Katlijn Vints, Ann Swijsen, Matthieu Moisse, et al. "Differentiation but not ALS mutations in FUS rewires motor neuron metabolism." Nature Communications 10, no. 1 (September 12, 2019). http://dx.doi.org/10.1038/s41467-019-12099-4.
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Abstract Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.
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Li, Qian, Yi Feng, Yingchao Xue, Xiping Zhan, Yi Fu, Gege Gui, Weiqiang Zhou, et al. "Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells." Molecular Neurodegeneration 17, no. 1 (January 10, 2022). http://dx.doi.org/10.1186/s13024-021-00510-y.
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Abstract Background Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. Methods We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. Results We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. Conclusions This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases. Graphical abstract
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Kim, Daehwan, Subin Kim, Ashley Sung, Neetika Patel, Nathan Wong, Michael J. Conboy, and Irina M. Conboy. "Autologous treatment for ALS with implication for broad neuroprotection." Translational Neurodegeneration 11, no. 1 (March 11, 2022). http://dx.doi.org/10.1186/s40035-022-00290-5.
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Abstract Background Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons (MNs), leading to paralysis, respiratory failure and death within 2–5 years of diagnosis. The exact mechanisms of sporadic ALS, which comprises 90% of all cases, remain unknown. In familial ALS, mutations in superoxide dismutase (SOD1) cause 10% of cases. Methods ALS patient-derived human-induced pluripotent stem cells (ALS hiPSCs, harboring the SOD1AV4 mutation), were differentiated to MNs (ALS-MNs). The neuroprotective effects of conditioned medium (CM) of hESCs (H9), wt hiPSCs (WTC-11) and the ALS iPSCs, on MN apoptosis and viability, formation and maintenance of neurites, mitochondrial activity and expression of inflammatory genes, were examined. For in vivo studies, 200 μl of CM from the ALS iPSCs (CS07 and CS053) was injected subcutaneously into the ALS model mice (transgenic for the human SOD1G93A mutation). Animal agility and strength, muscle innervation and mass, neurological score, onset of paralysis and lifespan of the ALS mice were assayed. After observing significant disease-modifying effects, the CM was characterized biochemically by fractionation, comparative proteomics, and epigenetic screens for the dependence on pluripotency. CM of fibroblasts that were differentiated from the wt hiPSCs lacked any neuroprotective activity and was used as a negative control throughout the studies. Results The secretome of PSCs including the ALS patient iPSCs was neuroprotective in the H2O2 model. In the model with pathogenic SOD1 mutation, ALS iPSC-CM attenuated all examined hallmarks of ALS pathology, rescued human ALS-MNs from denervation and death, restored mitochondrial health, and reduced the expression of inflammatory genes. The ALS iPSC-CM also improved neuro-muscular health and function, and delayed paralysis and morbidity in ALS mice. Compared side by side, cyclosporine (CsA), a mitochondrial membrane blocker that prevents the leakage of mitochondrial DNA, failed to avert the death of ALS-MNs, although CsA and ALS iPSC-CM equally stabilized MN mitochondria and attenuated inflammatory genes. Biochemical characterization, comparative proteomics, and epigenetic screen all suggested that it was the interactome of several key proteins from different fractions of PSC-CM that delivered the multifaceted neuroprotection. Conclusions This work introduces and mechanistically characterizes a new biologic for treating ALS and other complex neurodegenerative diseases.
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Krach, Florian, Emily C. Wheeler, Martin Regensburger, Tom Boerstler, Holger Wend, Anthony Q. Vu, Ruth Wang, et al. "Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis." Acta Neuropathologica, July 1, 2022. http://dx.doi.org/10.1007/s00401-022-02450-3.
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AbstractAmyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
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Massih, Bita, Alexander Veh, Maren Schenke, Simon Mungwa, Bettina Seeger, Bhuvaneish T. Selvaraj, Siddharthan Chandran, et al. "A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS." Frontiers in Cell and Developmental Biology 11 (February 14, 2023). http://dx.doi.org/10.3389/fcell.2023.996952.
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The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.
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Schulz, Carl, Marc D. Lemoine, Giulia Mearini, Jussi Koivumäki, Jascha Sani, Edzard Schwedhelm, Paulus Kirchhof, et al. "PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation." Circulation: Arrhythmia and Electrophysiology, February 10, 2023. http://dx.doi.org/10.1161/circep.122.011602.
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BACKGROUND: Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2 −/− ) in human induced pluripotent stem cell–derived atrial cardiomyocytes. METHODS: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell–derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell–derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. RESULTS: PITX2 −/− atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2 −/− than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2 −/− versus isogenic control; P <0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca 2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2 −/− versus isogenic control; P <0.0001), and maximum diastolic potential was more negative (−78.3±3.1 versus −69.7±0.6 mV, n=18/3 versus n=12/4, PITX2 −/− versus isogenic control; P =0.001), despite normal inward rectifier currents (both I K1 and I K,ACh ) and carbachol-induced shortening of action potential duration. CONCLUSIONS: Complete PITX2 deficiency in human induced pluripotent stem cell–derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.