scholarly journals Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1531 ◽  
Author(s):  
Kilian Mazaleyrat ◽  
Cherif Badja ◽  
Natacha Broucqsault ◽  
Raphaël Chevalier ◽  
Camille Laberthonnière ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Muscular Dystrophy ◽  
Pluripotent Stem Cells ◽  
Cell Biology ◽  
Disease Modeling ◽  
Muscle Fibers ◽  
Functional Evaluation ◽  
Skeletal Muscle Fibers ◽  
Human Ipscs

Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19–21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.

scholarly journals Directed Differentiation of Human Pluripotent Stem Cells toward Skeletal Myogenic Progenitors and Their Purification Using Surface Markers

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2746
Author(s):  
Nasa Xu ◽  
Jianbo Wu ◽  
Jose L. Ortiz-Vitali ◽  
Yong Li ◽  
Radbod Darabi
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Surface Markers ◽  
Gene Correction ◽  
Muscle Disorders

Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45–65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24−). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.

scholarly journals Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

2019 ◽  
Author(s):  
Richard L Carpenedo ◽  
Sarah Y Kwon ◽  
R Matthew Tanner ◽  
Julien Yockell-Lelièvre ◽  
Chandarong Choey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Biology ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Endoderm Differentiation ◽  
Prolonged Differentiation ◽  
Defined Culture

SummaryHuman pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

scholarly journals Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells

2020 ◽  
Author(s):  
Finn J. Hawkins ◽  
Shingo Suzuki ◽  
Mary Lou Beermann ◽  
Cristina Barillà ◽  
Ruobing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Basal Cell ◽  
Pluripotent Stem Cells ◽  
Airway Epithelium ◽  
Disease Modeling ◽  
Airway Epithelial ◽  
Human Ipscs ◽  
Lung Airways

SummaryThe derivation of self-renewing tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would shorten the time needed to engineer mature cell types in vitro and would have broad reaching implications for the field of regenerative medicine. Here we report the directed differentiation of human iPSCs into putative airway basal cells (“iBCs”), a population resembling the epithelial stem cell of lung airways. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato) we track and purify these cells over time, as they first emerge from iPSC-derived foregut endoderm as developmentally immature NKX2-1GFP+ lung progenitors which then augment a TP63 program during subsequent proximal airway epithelial patterning. These cells clonally proliferate, initially as NKX2-1GFP+/TP63tdTomato+ immature airway progenitors that lack expression of the adult basal cell surface marker NGFR. However, in response to primary basal cell medium, NKX2-1GFP+/ TP63tdTomato+ cells upregulate NGFR and display the molecular and functional phenotype of airway basal stem cells, including the capacity to clonally self-renew or undergo multilineage ciliated and secretory epithelial differentiation in air-liquid interface cultures. iBCs and their differentiated progeny recapitulate several fundamental physiologic features of normal primary airway epithelial cells and model perturbations that characterize acquired and genetic airway diseases. In an asthma model of mucus metaplasia, the inflammatory cytokine IL-13 induced an increase in MUC5AC+ cells similar to primary cells. CFTR-dependent chloride flux in airway epithelium generated from cystic fibrosis iBCs or their syngeneic CFTR-corrected controls exhibited a pattern consistent with the flux measured in primary diseased and normal human airway epithelium, respectively. Finally, multiciliated cells generated from an individual with primary ciliary dyskinesia recapitulated the ciliary beat and ultrastructural defects observed in the donor. Thus, we demonstrate the successful de novo generation of a tissue-resident stem cell-like population in vitro from iPSCs, an approach which should facilitate disease modeling and future regenerative therapies for a variety of diseases affecting the lung airways.

Human Pluripotent Stem Cells in Neurodegenerative Diseases: Potentials, Advances and Limitations

2020 ◽  
Vol 15 (2) ◽  
pp. 102-110 ◽  
Author(s):  
Tannaz Akbari Kolagar ◽  
Maryam Farzaneh ◽  
Negin Nikkar ◽  
Seyed Esmaeil Khoshnam
Keyword(s):  
Stem Cells ◽  
Neurodegenerative Diseases ◽  
Pluripotent Stem Cells ◽  
Neurodegenerative Disorders ◽  
Disease Modeling ◽  
Human Pluripotent Stem Cells ◽  
Neural Regeneration ◽  
Patient Specific ◽  
Human Escs ◽  
Human Ipscs

Neurodegenerative diseases are progressive and uncontrolled gradual loss of motor neurons function or death of neuron cells in the central nervous system (CNS) and the mechanisms underlying their progressive nature remain elusive. There is urgent need to investigate therapeutic strategies and novel treatments for neural regeneration in disorders like Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Currently, the development and identification of pluripotent stem cells enabling the acquisition of a large number of neural cells in order to improve cell recovery after neurodegenerative disorders. Pluripotent stem cells which consist of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their ability to indefinitely self-renew and the capacity to differentiate into different types of cells. The first human ESC lines were established from donated human embryos; while, because of a limited supply of donor embryos, human ESCs derivation remains ethically and politically controversial. Hence, hiPSCs-based therapies have been shown as an effective replacement for human ESCs without embryo destruction. Compared to the invasive methods for derivation of human ESCs, human iPSCs has opened possible to reprogram patient-specific cells by defined factors and with minimally invasive procedures. Human pluripotent stem cells are a good source for cell-based research, cell replacement therapies and disease modeling. To date, hundreds of human ESC and human iPSC lines have been generated with the aim of treating various neurodegenerative diseases. In this review, we have highlighted the recent potentials, advances, and limitations of human pluripotent stem cells for the treatment of neurodegenerative disorders.

scholarly journals The application of patient-derived induced pluripotent stem cells for modeling and treatment of Alzheimer’s disease

2019 ◽  
Vol 5 (1) ◽  
pp. 21-40 ◽  
Author(s):  
Fabin Han ◽  
Chuanguo Liu ◽  
Jin Huang ◽  
Juanli Chen ◽  
Chuanfei Wei ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Stem Cells ◽  
Human Brain ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Autologous Transplantation ◽  
Human Ipscs ◽  
Induced Pluripotent

Alzheimer’s disease (AD) is the most prevalent age-related neurodegenerative disease which is mainly caused by aggregated protein plaques in degenerating neurons of the brain. These aggregated protein plaques are mainly consisting of amyloid β (Aβ) fibrils and neurofibrillary tangles (NFTs) of phosphorylated tau protein. Even though the transgenic murine models can recapitulate some of the AD phenotypes, they are not the human cell models of AD. Recent breakthrough in somatic cell reprogramming made it available to use induced pluripotent stem cells (iPSCs) for patientspecific disease modeling and autologous transplantation therapy. Human iPSCs provide alternative ways to obtain specific human brain cells of AD patients to study the molecular mechanisms and therapeutic approaches for familial and sporadic forms of AD. After differentiation into neuronal cells, iPSCs have enabled the investigation of the complex aetiology and timescale over which AD develops in human brain. Here, we first go over the pathological process of and transgenic models of AD. Then we discuss the application of iPSC for disease model and cell transplantation. At last the challenges and future applications of iPSCs for AD will be summarized to propose cell-based approaches for the treatment of this devastating disorder.

scholarly journals Myogenesis modelled by human pluripotent stem cells uncovers Duchenne muscular dystrophy phenotypes prior to skeletal muscle commitment

2019 ◽  
Author(s):  
Virginie Mournetas ◽  
Emmanuelle Massouridès ◽  
Jean-Baptiste Dupont ◽  
Etienne Kornobis ◽  
Hélène Polvèche ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Pluripotent Stem Cells ◽  
Muscle Development ◽  
Mitochondrial Gene ◽  
Therapeutic Interventions ◽  
Disease Biomarkers ◽  
Induced Pluripotent

ABSTRACTDuchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5,000 male births. Symptoms appear in early childhood, with a diagnosis made around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise – even asymptomatically – is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets.In this study, we have used human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis, and compared their differentiation dynamics to that of healthy control cells by a comprehensive multi-omic analysis. Transcriptome and miRnome comparisons combined with protein analyses at 7 time points demonstrated that hiPSC differentiation 1) mimics described DMD phenotypes at the differentiation endpoint; and 2) homogeneously and robustly recapitulates key developmental steps - mesoderm, somite, skeletal muscle - which offers the possibility to explore dystrophin functions and find earlier disease biomarkers.Starting at the somite stage, mitochondrial gene dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of skeletal muscle cells that starts early during myogenesis. In sum, our data strongly argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin functions during muscle development.

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