scholarly journals Engineering skeletal muscle tissues with advanced maturity improves synapse formation with human induced pluripotent stem cell-derived motor neurons

2021 ◽  
Vol 5 (3) ◽  
pp. 036101
Author(s):  
Jeffrey W. Santoso ◽  
Xiling Li ◽  
Divya Gupta ◽  
Gio C. Suh ◽  
Eric Hendricks ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Synapse Formation ◽  
Induced Pluripotent Stem Cell ◽  
Muscle Tissues ◽  
Induced Pluripotent

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kimimasa Tobita ◽  
Jason S Tchao ◽  
Jong Kim ◽  
Bo Lin ◽  
Johnny Huard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Immature Myocardium

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

scholarly journals A Chemically Defined Common Medium for Culture of C2C12 Skeletal Muscle and Human Induced Pluripotent Stem Cell Derived Spinal Spheroids

2020 ◽  
Vol 13 (6) ◽  
pp. 605-619
Author(s):  
Rachel R. Besser ◽  
Annie C. Bowles ◽  
Ahmad Alassaf ◽  
Daniel Carbonero ◽  
Renata Maciel ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

scholarly journals Role of human induced pluripotent stem cell-derived spinal cord astrocytes in the functional maturation of motor neurons in a multielectrode array system

2019 ◽  
Author(s):  
Arens Taga ◽  
Raha Dastgheyb ◽  
Christa Habela ◽  
Jessica Joseph ◽  
Jean-Philippe Richard ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Stem Cell ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Time Dependent ◽  
Multielectrode Array ◽  
Electrophysiological Activity ◽  
Induced Pluripotent

AbstractThe ability to generate human induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system (CNS) biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-As are co-cultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to co-culture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron cross-talk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and co-culture with neurons influences astrocyte maturation as well. Taken together these data indicate that in a human in vitro spinal cord culture system, astrocytes alter hiPSC-MN maturation in a time-dependent and species specific manner and suggest a closer approximation of in vivo conditions.Main PointsWe developed a method for the co-culture of human iPSC-A/MN for multielectrode array recordings.The morphological, molecular, pharmacological, and electrophysiological characterization of the co-cultures suggests bidirectional maturation.

scholarly journals Human Induced Pluripotent Stem Cell-Derived TDP-43 Mutant Neurons Exhibit Consistent Functional Phenotypes Across Multiple Gene Edited Lines Despite Transcriptomic and Splicing Discrepancies

2021 ◽  
Vol 9 ◽  
Author(s):  
Alec S. T. Smith ◽  
Changho Chun ◽  
Jennifer Hesson ◽  
Julie Mathieu ◽  
Paul N. Valdmanis ◽  
...  
Keyword(s):  
Stem Cell ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Multiple Gene ◽  
Specific Effects ◽  
Induced Pluripotent ◽  
Careful Comparison ◽  
Lateral Sclerosis

Gene editing technologies hold great potential to enhance our ability to model inheritable neurodegenerative diseases. Specifically, engineering multiple amyotrophic lateral sclerosis (ALS) mutations into isogenic cell populations facilitates determination of whether different causal mutations cause pathology via shared mechanisms, and provides the capacity to separate these mechanisms from genotype-specific effects. As gene-edited, cell-based models of human disease become more commonplace, there is an urgent need to verify that these models constitute consistent and accurate representations of native biology. Here, commercially sourced, induced pluripotent stem cell-derived motor neurons from Cellular Dynamics International, edited to express the ALS-relevant mutations TDP-43M337V and TDP-43Q331K were compared with in-house derived lines engineered to express the TDP-43Q331K mutation within the WTC11 background. Our results highlight electrophysiological and mitochondrial deficits in these edited cells that correlate with patient-derived cells, suggesting a consistent cellular phenotype arising from TDP-43 mutation. However, significant differences in the transcriptomic profiles and splicing behavior of the edited cells underscores the need for careful comparison of multiple lines when attempting to use these cells as a means to better understand the onset and progression of ALS in humans.

scholarly journals Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair

2020 ◽  
Vol 29 (13) ◽  
pp. 2200-2217 ◽  
Author(s):  
Nidaa A Ababneh ◽  
Jakub Scaber ◽  
Rowan Flynn ◽  
Andrew Douglas ◽  
Paola Barbagallo ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Stem Cell ◽  
Genome Editing ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Dementia Patient ◽  
Homology Directed Repair ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

Abstract The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

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