scholarly journals Screening for axon regeneration promoting compounds with human iPSC-derived motor neurons

2021 ◽  
Author(s):  
Tammy Szu-Yu Ho ◽  
J. Tabitha Hees ◽  
Zhuqiu Xu ◽  
Riki Kawaguchi ◽  
Natalia P Biscola ◽  
...  
Keyword(s):  
Nerve Injury ◽  
Motor Neurons ◽  
Large Scale ◽  
Axon Regeneration ◽  
Induced Pluripotent Stem Cell ◽  
Screening Strategy ◽  
Therapeutic Interventions ◽  
Induced Pluripotent ◽  
Human Ipsc

CNS neurons do not regenerate after injury, leading to permanent functional deficits. Although sensory and motor neuron axons do regrow after peripheral nerve injury, functional outcome is limited due to the incomplete and slow regrowth. The lack of human-relevant assays suitable for large-scale drug screens has limited neuro-repair therapy discovery. To address this we developed a phenotypic screening strategy using human induced pluripotent stem cell-derived motor neurons to identify axon-regeneration promoting compounds and targets. The screens involve both re-plating human motor neurons on chondroitin sulfate proteoglycans and measuring regeneration responses to laser axotomy in spot cultures, and from them we identified multiple hits that promote injured axon regrowth. The top hit blebbistatin, a non-muscle myosin II inhibitor, accelerated axon regeneration and functional recovery after sciatic nerve injury in vivo. Human injury in a dish assays are suitable, therefore, to screen for therapeutic interventions that can induce or accelerate axon regeneration.

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.

Abstract 14104: Replacement of Infarct Myocardium by Large Scale-Expanded Human Induced Pluripotent Stem Cell-Derived Cardiac Cell-Sheet in a Porcine Chronic Myocardial Infarction Model

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Keitaro Domae ◽  
Shigeru Miyagawa ◽  
Satsuki Fukushima ◽  
Atsuhiro Saito ◽  
Yukiko Imanishi ◽  
...  
Keyword(s):  
Functional Recovery ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Cells ◽  
Cell Group ◽  
Small Scale ◽  
Cardiac Cell ◽  
Cell Sheets ◽  
Induced Pluripotent ◽  
Human Ipsc

Introduction: It has been shown that transplanted induced pluripotent stem cell (iPSC)-derived cardiac cells in the myocardial infarction (MI) heart synchronously contract with native myocardium to mechanically contribute to functional recovery in rodent models. We herein hypothesized that large scale cardiac cell-sheets generated by human iPSCs may induce a greater functional recovery than small scale ones after transplantation in chronic MI heart. Methods: Bioreactor-based three-dimensional suspension culture system was used for generating large scale-expanded human iPSC-derived cardiomyocytes, of which cardiac troponin T positivity was constantly 75-85%. Scaffold-free cell-sheets containing several cell number (1.0х10^6, 10^7, 10^8) were transplanted over the cardiac surface in porcine chronic MI heart (n=5 each). Tacrolimus and prednisolone were daily given in all pigs against xeno-transplantation-inducing immune reaction. Results: Echocardiographically, left ventricular systolic and diastolic dimensions were significantly decreasing and ejection fraction was significantly increasing in the 10^8 cell group. In addition, global myocardial structure was better preserved in the 10^8 cell group with presence of the graft in the infarct area macroscopically (Figure). Moreover, there were significantly less accumulation of interstitial fibrosis in the infarct-remote area and greater vascular density and expression of VEGF, bFGF, and SDF-1 in the infarct-border area in the 10^8 cell group than the other groups at 3 months after the transplantation. Conclusions: Large scale human iPSC-derived cardiac cells were engrafted in the infarct myocardium, showing substantial functional recovery in a porcine chronic MI heart, indicating that artificial cell-based myocardial replacement therapy may be achieved. In contrast, small scale cardiac cells induced modest functional recovery, suggesting paracrine mechanisms of this treatment.

scholarly journals Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis

Brain ◽  
2020 ◽  
Vol 143 (2) ◽  
pp. 430-440 ◽  
Author(s):  
Phillip Smethurst ◽  
Emmanuel Risse ◽  
Giulia E Tyzack ◽  
Jamie S Mitchell ◽  
Doaa M Taha ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Pathological Feature ◽  
Cell Type ◽  
Sporadic Als ◽  
Cell Type Specific ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by motor neuron loss, resulting in muscle wasting, paralysis and eventual death. A key pathological feature of ALS is cytoplasmically mislocalized and aggregated TDP-43 protein in >95% of cases, which is considered to have prion-like properties. Historical studies have predominantly focused on genetic forms of ALS, which represent ∼10% of cases, leaving the remaining 90% of sporadic ALS relatively understudied. Additionally, the role of astrocytes in ALS and their relationship with TDP-43 pathology is also not currently well understood. We have therefore used highly enriched human induced pluripotent stem cell (iPSC)-derived motor neurons and astrocytes to model early cell type-specific features of sporadic ALS. We first demonstrate seeded aggregation of TDP-43 by exposing human iPSC-derived motor neurons to serially passaged sporadic ALS post-mortem tissue (spALS) extracts. Next, we show that human iPSC-derived motor neurons are more vulnerable to TDP-43 aggregation and toxicity compared with their astrocyte counterparts. We demonstrate that these TDP-43 aggregates can more readily propagate from motor neurons into astrocytes in co-culture paradigms. We next found that astrocytes are neuroprotective to seeded aggregation within motor neurons by reducing (mislocalized) cytoplasmic TDP-43, TDP-43 aggregation and cell toxicity. Furthermore, we detected TDP-43 oligomers in these spALS spinal cord extracts, and as such demonstrated that highly purified recombinant TDP-43 oligomers can reproduce this observed cell-type specific toxicity, providing further support to a protein oligomer-mediated toxicity hypothesis in ALS. In summary, we have developed a human, clinically relevant, and cell-type specific modelling platform that recapitulates key aspects of sporadic ALS and uncovers both an initial neuroprotective role for astrocytes and the cell type-specific toxic effect of TDP-43 oligomers.

scholarly journals Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell–Derived Endothelial Cells

2017 ◽  
Vol 37 (11) ◽  
pp. 2014-2025 ◽  
Author(s):  
Yang Lin ◽  
Chang-Hyun Gil ◽  
Mervin C. Yoder
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Induced Pluripotent ◽  
Human Ipsc

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony–forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols.

scholarly journals A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and in vivo tracking of therapeutic cells

2018 ◽  
Vol 373 (1750) ◽  
pp. 20170219 ◽  
Author(s):  
Martha Lopez-Yrigoyen ◽  
Antonella Fidanza ◽  
Luca Cassetta ◽  
Richard A. Axton ◽  
A. Helen Taylor ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Efficient Production ◽  
Ipsc Line ◽  
Fluorescent Reporter ◽  
Surface Marker Expression ◽  
Haematopoietic Cells ◽  
Induced Pluripotent ◽  
High Level ◽  
Human Ipsc

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Continuation of fast axonal transport in regenerating axons in vitro

1979 ◽  
Vol 57 (11) ◽  
pp. 1251-1255
Author(s):  
M. A. Bisby ◽  
C. E. Hilton
Keyword(s):  
Sciatic Nerve ◽  
Vagus Nerve ◽  
Axonal Transport ◽  
Nerve Injury ◽  
Axon Regeneration ◽  
Free Medium ◽  
Fast Axonal Transport ◽  
Sensory Axons

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

scholarly journals Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Peinkofer ◽  
Martina Maass ◽  
Kurt Pfannkuche ◽  
Agapios Sachinidis ◽  
Stephan Baldus ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Developmental Stage ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

scholarly journals Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes

2017 ◽  
Vol 312 (6) ◽  
pp. H1144-H1153 ◽  
Author(s):  
Sam Chai ◽  
Xiaoping Wan ◽  
Drew M. Nassal ◽  
Haiyan Liu ◽  
Christine S. Moravec ◽  
...  
Keyword(s):  
Action Potential ◽  
Expression Profile ◽  
Cardiac Electrophysiology ◽  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Induced Pluripotent ◽  
Interfering Rna ◽  
Human Ipsc ◽  
Physiological Systems

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

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