scholarly journals Engineered developmental niche enables predictive phenotypic screening in human dystrophic cardiomyopathy

2018 ◽  
Author(s):  
Jesse R. Macadangdang ◽  
Jason W. Miklas ◽  
Alec S.T. Smith ◽  
Eunpyo Choi ◽  
Winnie Leung ◽  
...  
Keyword(s):  
Microelectrode Array ◽  
Disease Modeling ◽  
Contractile Function ◽  
Calcium Content ◽  
Cytosolic Calcium ◽  
Biochemical Properties ◽  
Directed Differentiation ◽  
Dystrophic Cardiomyopathy ◽  
Mitochondrial Capacity

ABSTRACTDirected differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes typically produces cells with structural, functional, and biochemical properties that most closely resemble those present in the fetal heart. Here we establish an in vitro engineered developmental cardiac niche to produce matured hPSC-derived cardiomyocytes (hPSC-CMs) with enhanced sarcomere development, electrophysiology, contractile function, mitochondrial capacity, and a more mature transcriptome. When this developmental cardiac niche was applied to dystrophin mutant hPSC-CMs, a robust disease phenotype emerged, which was not observed in non-matured diseased hPSC-CMs. Matured dystrophin mutant hPSC-CMs exhibited a greater propensity for arrhythmia as measured via beat rate variability, most likely due to higher resting cytosolic calcium content. Using a custom nanopatterned microelectrode array platform to screen functional output in hPSC-CMs exposed to our engineered developmental cardiac niche, we identified calcium channel blocker, nitrendipine, mitigated hPSC-CM arrhythmogenic behavior and correctly identified sildenafil as a false positive. Taken together, we demonstrate our developmental cardiac niche platform enables robust hPSC-CM maturation allowing for more accurate disease modeling and predictive drug screening.

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals Modeling endodermal organ development and diseases using human pluripotent stem cell-derived organoids

2020 ◽  
Vol 12 (8) ◽  
pp. 580-592
Author(s):  
Fong Cheng Pan ◽  
Todd Evans ◽  
Shuibing Chen
Keyword(s):  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
State Of The Art ◽  
Directed Differentiation ◽  
Organ Development ◽  
Human Pluripotent Stem Cell ◽  
Human Organ ◽  
Current State

Abstract Recent advances in development of protocols for directed differentiation from human pluripotent stem cells (hPSCs) to defined lineages, in combination with 3D organoid technology, have facilitated the generation of various endoderm-derived organoids for in vitro modeling of human gastrointestinal development and associated diseases. In this review, we discuss current state-of-the-art strategies for generating hPSC-derived endodermal organoids including stomach, liver, pancreatic, small intestine, and colonic organoids. We also review the advantages of using this system to model various human diseases and evaluate the shortcomings of this technology. Finally, we emphasize how other technologies, such as genome editing and bioengineering, can be incorporated into the 3D hPSC-organoid models to generate even more robust and powerful platforms for understanding human organ development and disease modeling.

scholarly journals Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

2017 ◽  
Vol 2017 ◽  
pp. 1-18 ◽  
Author(s):  
Divya S. Varghese ◽  
Shama Parween ◽  
Mustafa T. Ardah ◽  
Bright Starling Emerald ◽  
Suraiya A. Ansari
Keyword(s):  
Cell Viability ◽  
Disease Modeling ◽  
Culture Media ◽  
Embryonic Stem ◽  
Toxicity Testing ◽  
Cell Types ◽  
Specific Cell ◽  
Directed Differentiation ◽  
Embryonic Neurogenesis

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results.

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 Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sridhar Selvaraj ◽  
Ricardo Mondragon-Gonzalez ◽  
Bin Xu ◽  
Alessandro Magli ◽  
Hyunkee Kim ◽  
...  
Keyword(s):  
Disease Modeling ◽  
Contractile Function ◽  
Myogenic Differentiation ◽  
Three Dimensional ◽  
Electrical Stimulus ◽  
Chromatin Accessibility ◽  
Myosin Heavy Chain Isoforms ◽  
Disease Phenotypes ◽  
Small Molecule Screen

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells.

scholarly journals Characterization of directed differentiation by high-throughput single-cell RNA-Seq

2014 ◽  
Author(s):  
Magali Soumillon ◽  
Davide Cacchiarelli ◽  
Stefan Semrau ◽  
Alexander van Oudenaarden ◽  
Tarjei S Mikkelsen
Keyword(s):  
Gene Expression ◽  
Disease Modeling ◽  
Single Cells ◽  
Adipogenic Differentiation ◽  
Digital Gene Expression ◽  
Multiple Time ◽  
Directed Differentiation ◽  
Time Points

Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems is complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 12,832 cells collected at multiple time points during directed adipogenic differentiation of human adipose-derived stem/stromal cells in vitro. The resulting data reveal the major axes of cell-to-cell variation within and between time points, and an inverse relationship between inflammatory gene expression and lipid accumulation across cells from a single donor.

Abstract 156: Hydrogel Mattress, an in vitro Platform to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Tromondae K Feaster ◽  
Charles H Williams ◽  
Adrian G Cadar ◽  
Young W Chun ◽  
Lili Wang ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Disease Modeling ◽  
Contractile Function ◽  
Traction Force ◽  
Induced Pluripotent Stem Cell ◽  
Contractile Properties ◽  
Calcium Handling ◽  
Cell Shortening ◽  
Induced Pluripotent

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

scholarly journals An efficient method to generate kidney organoids at the air-liquid interface

2021 ◽  
Vol 8 (2) ◽  
pp. e150
Author(s):  
Ashwani Kumar Gupta ◽  
David Z. Ivancic ◽  
Bilal A. Naved ◽  
Jason A. Wertheim ◽  
Leif Oxburgh
Keyword(s):  
Disease Modeling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Cell ◽  
Directed Differentiation ◽  
Chemical Screening ◽  
Air Liquid Interface ◽  
Kidney Organoids

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

scholarly journals Human Cardiac Fibroblast Number and Activation State Modulate Electromechanical Function of hiPSC-Cardiomyocytes in Engineered Myocardium

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Cassady E. Rupert ◽  
Tae Yun Kim ◽  
Bum-Rak Choi ◽  
Kareen L. K. Coulombe
Keyword(s):  
Cardiac Tissue ◽  
Disease Modeling ◽  
Contractile Function ◽  
Cardiac Fibroblasts ◽  
Cell Number ◽  
Cardiac Fibroblast ◽  
Cellular Interactions ◽  
Excitation Rate ◽  
Cardiac Tissues

Cardiac tissue engineering using hiPSC-derived cardiomyocytes is a promising avenue for cardiovascular regeneration, pharmaceutical drug development, cardiotoxicity evaluation, and disease modeling. Limitations to these applications still exist due in part to the need for more robust structural support, organization, and electromechanical function of engineered cardiac tissues. It is well accepted that heterotypic cellular interactions impact the phenotype of cardiomyocytes. The current study evaluates the functional effects of coculturing adult human cardiac fibroblasts (hCFs) in 3D engineered tissues on excitation and contraction with the goal of recapitulating healthy, nonarrhythmogenic myocardium in vitro. A small population (5% of total cell number) of hCFs in tissues improves tissue formation, material properties, and contractile function. However, two perturbations to the hCF population create disease-like phenotypes in engineered cardiac tissues. First, increasing the percentage of hCFs to 15% resulted in tissues with increased ectopic activity and spontaneous excitation rate. Second, hCFs undergo myofibroblast activation in traditional two-dimensional culture, and this altered phenotype ablated the functional benefits of hCFs when incorporated into engineered cardiac tissues. Taken together, the results of this study demonstrate that human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes and that a low percentage of quiescent hCFs are a valuable cell source to advance a healthy electromechanical response of engineered cardiac tissue for regenerative medicine applications.

scholarly journals Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation In Vitro

2012 ◽  
Vol 17 (9) ◽  
pp. 1192-1203 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Kenichi Imahashi ◽  
Hiroshi Sawada ◽  
Hatsue Furukawa ◽  
Kenji Takami
Keyword(s):  
Stem Cell ◽  
Sarcoplasmic Reticulum ◽  
Pluripotent Stem Cell ◽  
Microelectrode Array ◽  
Contractile Function ◽  
Induced Pluripotent Stem Cell ◽  
Channel Blockers ◽  
Electrophysiological Properties ◽  
Induced Pluripotent

Human-induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs) at different stages (approximate days 30, 60, and 90) were used to determine the appropriate stage for functional and morphological assessment of drug effects in vitro. The hiPS-CMs had spontaneous beating activity, and β-adrenergic function was comparable in all stages of differentiation. Microelectrode array analyses using ion channel blockers indicated that the electrophysiological properties of these ion channels were comparable at all differentiation stages. Ultrastructural analysis using electron microscopy showed that myofibrillar structures at days 60 and 90 were similarly distributed and more mature than that at day 30. Analysis of motion vectors in contracting cells showed that the velocity of contraction was the highest at day 90 and was the most mature among the three stages. Gene expression analysis demonstrated that expression of some genes related to myofilament and sarcoplasmic reticulum increased with maturation of morphological and contractile properties. In conclusion, day 30 cardiomyocytes are useful for basic screening such as the assessment of electrophysiological properties, and days 60 and 90 are the appropriate differentiation stage for morphological assays. For the assay of contractile function associated with subcellular components such as sarcoplasmic reticulum, day 90 cardiomyocytes are the most suitable.

Sign in / Sign up

Export Citation Format

Share Document