Agnieszka Pastula
  • Faculty, Uniwerstet Jagiellonski
Research fields
  • Cancer Biology, Cell Biology, Molecular Biology, Stem Cell
Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
Authors:  Megan D. Schertzer, McKenzie M. Murvin and J. Mauro Calabrese, date: 10/05/2020, view: 4003, Q&A: 0
Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular abundance, especially for lncRNAs that accumulate in focused subcellular regions. Calibrated RT-qPCR may return inaccurate estimates of abundance because individual PCR amplicons spaced across the length of a transcript can vary in their efficiency of reverse transcription. Here, we describe a sequencing-based approach that is straightforward, orthogonal to smFISH and RT-qPCR, and can be used to approximate the intracellular abundance for most expressed long RNAs (lncRNAs and mRNAs) in a cell type of interest. Firstly, the average weight of total RNA per cell for the cell type of interest is estimated by replicate rounds of RNA purification from a known number of cells. Secondly, an rRNA-depletion RNA-Seq protocol is performed after adding spike-in control RNAs to a known quantity of total cellular RNA. Lastly, by comparing read counts per transcript to a standard curve derived from the spiked-in RNAs, the intracellular abundance for each transcript is estimated. The sequencing-based approach provides a powerful complement to existing methods, particularly in situations where it is desirable to quantify the abundance of multiple lncRNAs and/or mRNAs simultaneously.
In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)
Authors:  Agnieszka D’Antonio-Chronowska, Matteo D’Antonio and Kelly A. Frazer, date: 12/20/2019, view: 6429, Q&A: 0
Induced Pluripotent Stem Cells (iPSCs) serve as an excellent model system for studying the molecular underpinnings of tissue development. Human iPSC-derived retinal pigment epithelium (iPSC-RPE) cells have fetal-like molecular profiles. Hence, biobanks like iPSCORE, which contain iPSCs generated from hundreds of individuals, are an invaluable resource for examining how common genetic variants exert their effects during RPE development resulting in individuals having different propensities to develop Age-related Macular Degeneration (AMD) as adults. Here, we present an optimized, cost-effective and highly reproducible protocol for derivation of human iPSC-RPE cells using small molecules under serum-free condition and for their quality control using flow cytometry and immunofluorescence. While most previous protocols have required laborious manual selection to enrich for iPSC-RPE cells, our protocol uses whole culture passaging and yields a large number of iPSC-RPE cells with high purity (88-98.1% ZO-1 and MiTF double positive cells). The simplicity and robustness of this protocol would enable its adaption for high-throughput applications involving the generation of iPSC-RPE samples from hundreds of individuals.
Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments
Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications.

This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.
Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
Authors:  Anthony Flamier, Andrea Barabino and Gilbert Bernier, date: 07/20/2016, view: 9820, Q&A: 1
Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.
Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro
Authors:  Per Anderson, Ana Belén Carrillo-Gálvez and Francisco Martín, date: 11/05/2015, view: 10554, Q&A: 0
Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.
Genome-Wide siRNA Screen for Anti-Cancer Drug Resistance in Adherent Cell Lines
Authors:  Elza C. de Bruin, Ming Jiang, Michael Howell and Julian Downward, date: 05/20/2015, view: 12338, Q&A: 0
The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the human siGENOME library from Dharmacon. In this protocol, we focus on resistance to treatment with the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) erlotinib in the lung cancer cell line PC9, which is exquisitely sensitive to EGFR-TKIs (de Bruin et al., 2014). This protocol can be used for other cell lines and other drug treatments, as we expand in the Notes below.
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