Abstract 397: Differentiation and Maturation of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes in Nanopatterned Cell Culture

Background: Human induced pluripotent stem cells (hiPSCs) are widely used in studies of developmental and regenerative biomedicine involving various cell types, including cardiomyocytes (CMs). Understanding the cellular and molecular processes during hiPSC-CM differentiation and maturation will be crucial to develop heart regeneration therapies. In addition, while bioengineered cellular cues have been shown to modulate cellular morphology and phenotypes, little is known regarding their effects on molecular and functional maturity of hiPSC-CMs. Aims: To determine the expression of cardiac and cell cycle genes and the electrophysiological properties of hiPSC-CMs during their differentiation and maturation on unpatterned (Flat) or isotropic/nanopatterned (Nano) cell culture surface. Methods and Results: Using small molecules, WTC-11 hiPSC were differentiated into CMs efficiently (92.5% TNNT2 + ; 95% NKX2-5 + ). The resultant hiPSC-CMs were re-plated in Flat or Nano surfaces and harvested at different time points. Cardiac genes Myh7, Tnnt2, Serca2a, Ryr2, Cacna1c , and Kcnj2 gradually and significantly increased during differentiation; this was accompanied by reduced expression of cell cycle genes. While myofilament genes expressions were similar between Nano and Flat cultured hiPSC-CMs, ion channel genes Scn5a, Cacna1c , and Kcnj2 were significantly higher in Nano group, suggesting that Nano cultured CMs were more matured. In addition, fewer hiPSC-CMs were proliferative (EdU + ) in 2-week Nano group compared to Flat group. This was associated with decreased expression of active cell cycle genes Ccne1, Cdk4, Cdk14, Ki67 and Plk1 in Nano 2-week CMs. Micro-electrode array (MEA) analysis demonstrated that Beat Period, Spike Amplitude, and Field Potential Duration were increased in the Nano group. Fluo-4 Ca 2+ imaging assay revealed improved Ca 2+ transition activities in isotropically cultured hiPSC-CMs. Conclusion: These results demonstrate a significant upregulation of cardiac genes along with a down-regulation of cell cycle genes during the differentiation and maturation of hiPSC-CM on Nano surfaces. Bioengineered nanotopographically patterned substrates promoted the maturation and electrophysiological functions of hiPSC-CMs.

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An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry

10.1101/2021.09.30.462679 ◽

2021 ◽

Author(s):

Anthony Duchesne ◽

Jing Dong ◽

Andrew N. Bayne ◽

Nguyen-Vi Mohamed ◽

Wei Yi ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Culture ◽

Protein Turnover ◽

Isotope Labeling ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Turnover Rates ◽

Cell Type Specificity ◽

Induced Pluripotent

AbstractPatient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinson’s disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

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Faculty Opinions recommendation of Small molecule screening in human induced pluripotent stem cell-derived terminal cell types.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718215280.793488931 ◽

2013 ◽

Author(s):

John Lowe

Keyword(s):

Stem Cell ◽

Small Molecule ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Terminal Cell ◽

Small Molecule Screening ◽

Induced Pluripotent

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Nanofibrous Matrix of Defined Composition Sustains Human Induced Pluripotent Stem Cell Culture

ACS Applied Bio Materials ◽

10.1021/acsabm.0c00425 ◽

2021 ◽

Vol 4 (4) ◽

pp. 3035-3040

Author(s):

Sara Borrego-González ◽

Berta de la Cerda ◽

Francisco J. Díaz-Corrales ◽

Aránzazu Díaz-Cuenca

Keyword(s):

Cell Culture ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Stem Cell Culture ◽

Induced Pluripotent

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A Temperature-Controlled Patch Clamp Platform Demonstrated on Jurkat T Lymphocytes and Human Induced Pluripotent Stem Cell-Derived Neurons

Bioengineering ◽

10.3390/bioengineering7020046 ◽

2020 ◽

Vol 7 (2) ◽

pp. 46 ◽

Cited By ~ 1

Author(s):

Jann Harberts ◽

Max Kusch ◽

John O’Sullivan ◽

Robert Zierold ◽

Robert H. Blick

Keyword(s):

Stem Cell ◽

T Lymphocytes ◽

Patch Clamp ◽

Pluripotent Stem Cell ◽

Room Temperature ◽

Standard Procedure ◽

Induced Pluripotent Stem Cell ◽

Electrophysiological Properties ◽

Temperature Controlled ◽

Induced Pluripotent

Though patch clamping at room temperature is a widely disseminated standard procedure in the electrophysiological community, it does not represent the biological system in mammals at around 37 °C. In order to better mimic the natural environment in electrophysiological studies, we present a custom-built, temperature-controlled patch clamp platform for upright microscopes, which can easily be adapted to any upright patch clamp setup independently, whether commercially available or home built. Our setup can both cool and heat the platform having only small temperature variations of less than 0.5 °C. We demonstrate our setup with patch clamp measurements at 36 °C on Jurkat T lymphocytes and human induced pluripotent stem cell-derived neurons. Passive membrane parameters and characteristic electrophysiological properties, such as the gating properties of voltage-gated ion channels and the firing of action potentials, are compared to measurements at room temperature. We observe that many processes that are not explicitly considered as temperature dependent show changes with temperature. Thus, we believe in the need of a temperature control in patch clamp measurements if improved physiological conditions are required. Furthermore, we advise researchers to only compare electrophysiological results directly that have been measured at similar temperatures since small variations in cellular properties might be caused by temperature alterations.

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

10.1101/2020.04.10.036400 ◽

2020 ◽

Cited By ~ 1

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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CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

10.1101/2021.05.24.445360 ◽

2021 ◽

Author(s):

Zhengyu Ouyang ◽

Nathanael Bourgeois ◽

Eugenia Lyashenko ◽

Paige Cundiff ◽

Patrick F Cullen ◽

...

Keyword(s):

Single Cell ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Model Systems ◽

Rna Seq ◽

Cell Type ◽

Fine Grained ◽

Single Nucleus ◽

Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

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