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

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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Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210696 ◽

2021 ◽

pp. 1-20

Author(s):

James S. Novak ◽

Rita Spathis ◽

Utkarsh J. Dang ◽

Alyson A. Fiorillo ◽

Ravi Hindupur ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Expression ◽

Protein Turnover ◽

Isotope Labeling ◽

Exon Skipping ◽

Mdx Mice ◽

Wild Type ◽

Gene Correction ◽

Dystrophin Protein

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystroglycan complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

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Modeling Intestinal Epithelial Response to Interferon-γ in Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids

International Journal of Molecular Sciences ◽

10.3390/ijms22010288 ◽

2020 ◽

Vol 22 (1) ◽

pp. 288

Author(s):

Michael J. Workman ◽

Elissa Troisi ◽

Stephan R. Targan ◽

Clive N. Svendsen ◽

Robert J. Barrett

Keyword(s):

Interferon Gamma ◽

Adherens Junction ◽

Induced Pluripotent Stem Cell ◽

Enrichment Analysis ◽

Cell Types ◽

Gene Set Enrichment Analysis ◽

Intestinal Organoids ◽

Ifn Γ ◽

Induced Pluripotent

Human intestinal organoids (HIOs) are increasingly being used to model intestinal responses to various stimuli, yet few studies have confirmed the fidelity of this modeling system. Given that the interferon-gamma (IFN-γ) response has been well characterized in various other cell types, our goal was to characterize the response to IFN-γ in HIOs derived from induced pluripotent stem cells (iPSCs). To achieve this, iPSCs were directed to form HIOs and subsequently treated with IFN-γ. Our results demonstrate that IFN-γ phosphorylates STAT1 but has little effect on the expression or localization of tight and adherens junction proteins in HIOs. However, transcriptomic profiling by microarray revealed numerous upregulated genes such as IDO1, GBP1, CXCL9, CXCL10 and CXCL11, which have previously been shown to be upregulated in other cell types in response to IFN-γ. Notably, “Response to Interferon Gamma” was determined to be one of the most significantly upregulated gene sets in IFN-γ-treated HIOs using gene set enrichment analysis. Interestingly, similar genes and pathways were upregulated in publicly available datasets contrasting the gene expression of in vivo biopsy tissue from patients with IBD against healthy controls. These data confirm that the iPSC-derived HIO modeling system represents an appropriate platform to evaluate the effects of various stimuli and specific environmental factors responsible for the alterations in the intestinal epithelium seen in various gastrointestinal conditions such as inflammatory bowel disease.

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

Circulation Research ◽

10.1161/res.127.suppl_1.397 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Yiqiang Zhang ◽

Jong-Seob Choi ◽

Alec Smith ◽

Robb MacLellan ◽

Deok-Ho Kim ◽

...

Keyword(s):

Cell Cycle ◽

Cell Culture ◽

Field Potential ◽

Electrode Array ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Electrophysiological Properties ◽

Cell Cycle Genes ◽

Induced Pluripotent ◽

Regulation Of Cell Cycle

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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A mass spectrometry workflow for measuring protein turnover rates in vivo

Nature Protocols ◽

10.1038/s41596-019-0222-y ◽

2019 ◽

Vol 14 (12) ◽

pp. 3333-3365 ◽

Cited By ~ 3

Author(s):

Mihai Alevra ◽

Sunit Mandad ◽

Till Ischebeck ◽

Henning Urlaub ◽

Silvio O. Rizzoli ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Turnover ◽

Turnover Rates

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An atlas of protein turnover rates in mouse tissues

Nature Communications ◽

10.1038/s41467-021-26842-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zach Rolfs ◽

Brian L. Frey ◽

Xudong Shi ◽

Yoshitaka Kawai ◽

Lloyd M. Smith ◽

...

Keyword(s):

Protein Turnover ◽

Isotope Labeling ◽

Turnover Rates ◽

Visualization Software ◽

Degradation Rates ◽

Mouse Tissues ◽

Related Drug ◽

Synthesis And Degradation ◽

Mammalian Protein

AbstractProtein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins—including those of the extracellular matrix—in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.

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High-resolution transcriptional landscape of xeno-free human induced pluripotent stem cell-derived cerebellar organoids

Scientific Reports ◽

10.1038/s41598-021-91846-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samuel Nayler ◽

Devika Agarwal ◽

Fabiola Curion ◽

Rory Bowden ◽

Esther B. E. Becker

Keyword(s):

Three Dimensional ◽

Growth Dynamics ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Cerebellar Development ◽

Disease Genes ◽

Complex Processes ◽

Cerebellar Cell ◽

Induced Pluripotent

AbstractCurrent protocols for producing cerebellar neurons from human pluripotent stem cells (hPSCs) often rely on animal co-culture and mostly exist as monolayers, limiting their capability to recapitulate the complex processes in the developing cerebellum. Here, we employed a robust method, without the need for mouse co-culture to generate three-dimensional cerebellar organoids from hPSCs that display hallmarks of in vivo cerebellar development. Single-cell profiling followed by comparison to human and mouse cerebellar atlases revealed the presence and maturity of transcriptionally distinct populations encompassing major cerebellar cell types. Encapsulation with Matrigel aimed to provide more physiologically-relevant conditions through recapitulation of basement-membrane signalling, influenced both growth dynamics and cellular composition of the organoids, altering developmentally relevant gene expression programmes. We identified enrichment of cerebellar disease genes in distinct cell populations in the hPSC-derived cerebellar organoids. These findings ascertain xeno-free human cerebellar organoids as a unique model to gain insight into cerebellar development and its associated disorders.

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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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Stem Cell Cure for Kidney Injury: Therapy to Solve Most Complex Issues in Renal System

Journal of Regenerative Biology and Medicine ◽

10.37191/mapsci-2582-385x-2(4)-038 ◽

2020 ◽

Author(s):

Prithiv K R Kumar

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Kidney Injury ◽

Cell Types ◽

Tissue Remodelling ◽

Major Health Problem ◽

In Vivo Experiments ◽

Renal Regeneration ◽

Induced Pluripotent

Renal failure is a major health problem. The mortality rate remain high despite of several therapies. The most complex of the renal issues are solved through stem cells. In this review, different mechanism for cure of chronic kidney injury along with cell engraftment incorporated into renal structures will be analysed. Paracrine activities of embryonic or induced Pluripotent stem cells are explored on the basis of stem cell-induced kidney regeneration. Several experiments have been conducted to advance stem cells to ensure the restoration of renal functions. More vigour and organised protocols for delivering stem cells is a possibility for advancement in treatment of renal disease. Also there is a need for pressing therapies to replicate the tissue remodelling and cellular repair processes suitable for renal organs. Stem cells are the undifferentiated cells that have the ability to multiply into several cell types. In vivo experiments on animal’s stem cells have shown significant improvements in the renal regeneration and functions of organs. Nevertheless more studies show several improvements in the kidney repair due to stem cell regeneration.

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