Interstitial lung diseases (ILD) are a group of rare lung diseases with severe outcomes. The COST Innovator Grant aims to establish a first-of-a-kind open-access Biorepository of patient-derived ...induced pluripotent stem cells (iPSC) and to train researchers in the skills required to generate a robust preclinical model of ILD using these cells. This study aims to describe and evaluate the effectiveness of a training course designed to train researchers in iPSC techniques to model ILD.
74 researchers, physicians and stakeholders attended the training course in Dublin in May 2022 with 31 trainees receiving teaching in practical iPSC culturing skills. The training course learners were divided into the Hands-on (16 trainees) and Observer groups (15 trainees), with the Observers attending a supervised live-streamed experience of the laboratories skills directly delivered to the Hands-on group. All participants were asked to participate in an evaluation to analyse their satisfaction and knowledge gained during the Training Course, with means compared using t-tests.
The gender balance in both groups was predominantly females (77.4%). The Hands-on group consisted mainly of researchers (75%), whereas all participants of the Observer group described themselves as clinicians. All participants in the Hands-on group were at least very satisfied with the training course compared to 70% of the participants in the Observer group. The knowledge assessment showed that the Hands-on group retained significantly more knowledge of iPSC characteristics and culturing techniques compared to the Observers (* < 0.05; p = 0.0457). A comprehensive learning video detailing iPSC culturing techniques was produced and is included with this manuscript.
The majority of participants were highly or very satisfied with the training course and retained significant knowledge about iPSC characteristics and culturing techniques after attending the training course. Overall, our findings demonstrate the feasibility of running hybrid Hands-on and Observer teaching events and underscore the importance of this type of training programme to appeal to a broad spectrum of interested clinicians and researchers particularly in rare disease. The long-term implications of this type of training event requires further study to determine its efficacy and impact on adoption of iPSC disease modelling techniques in participants' laboratories.
Background There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. ...However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). Methods We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. Results The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. Conclusions The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications. Keywords: Organoids, Hematopoiesis, hiPSC, Macrophages, Up-scaling, Bioreactor, Drug screening, Cell manufacturing
Alveolar macrophages (AMs) represent crucial immune cells in the bronchioalveolar space of the lung. Given the important role in the host defense machinery and lung tissue homeostasis, AMs have been ...linked to a variety of diseases and thus represent a promising target cell type for novel therapies. The emerging importance of AM underlines the necessity to isolate and/or generate proper cellular models, which facilitate basic biology and translational science. As of yet, most studies focus on the derivation of AM from the murine system. This chapter introduces the use of human-induced pluripotent stem cell (iPSC)-derived primitive macrophages, which can be further matured towards an AM-like phenotype upon intra-pulmonary transfer into mice. We will give a brief overview on the generation of primitive iPSC-derived macrophages, which is followed by a detailed, step-by-step description of the intra-pulmonary transfer of cells and the follow-up procedures needed to isolate the iPSC-derived, AM-like cells from the lungs post-transfer. The chapter provides an alternative approach to derive human AM-like cells, which can be used to study human AM biology and to investigate novel therapeutic interventions using primitive macrophages from iPSC.
Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient ...bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.
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