Derivation and Functional Analysis of Patient-Specific Induced Pluripotent Stem Cells as an In Vitro Model of Chronic Granulomatous Disease

Institute of Genetic Medicine, Newcastle University, Newcastle, UK.
Stem Cells (Impact Factor: 6.52). 04/2012; 30(4):599-611. DOI: 10.1002/stem.1053
Source: PubMed


Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47phox and gp91phox) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47phox or gp91phox mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy. Stem Cells
2012; 30:599–611

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Available from: Gabriele Saretzki
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    • "To assess whether PSC-MDM resemble b-MDM functionally, PCS-MDM were assayed for their ability to secrete cytokines and chemokines upon activation. Resting PSC-MDM (Figure 5, top panel) had a remarkably similar profile of cytokine production as b-MDM (b-MDM data shown in Jiang et al. [18], using exactly the same assay). As expected, cytokine secretion was upregulated upon classical activation (Figure 5, middle panel), but distinct from expression profiles of alternatively activated PSC-MDM (Figure 5, bottom panel) and from expression profiles reported for other cell types, such as T-cells [19], [20], [21]. "
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    ABSTRACT: Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×10(7) monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14(+), CD16(low), CD163(+)). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.
    Preview · Article · Aug 2013 · PLoS ONE
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    • "Furthermore, a number of disease-associated iPSCs generated from patients with immunological disorders have been reported. [15], [31]–[34] Because patient- or disease-specific iPS cells will be an important resource for unraveling human immunological disorders, a robust and simple hematopoietic differentiation system that can reliably mimic in vivo hematopoiesis is necessary for this purpose. Our simple and robust protocol to produce monocytic cells is therefore expected to be useful for regenerative medicine and studies of immunological disorders. "
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    ABSTRACT: Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10(6)±0.3×10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.
    Full-text · Article · Apr 2013 · PLoS ONE
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    ABSTRACT: Copyright: © 2012 Young W, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The breakthrough development of induced pluripotent stem cell (iPSC) technology is not only revolutionizing basic stem cell science but is also spurring efforts to reprogram one somatic cell type directly into another. Induced pluripotent stem cells provide scientists with a self-renewing and, thus, unlimited, source of pluripotent cells for targeted differentiation, in principle, into the entire range of cell types found in the body. Therefore, iPSC technology and the increasingly refined abilities to differentiate iPSCs into disease-relevant mature cells has far reaching implications for understanding disease etiology and promoting drug discovery and other advances in regenerative medicine. In this review, we summarize the latest progress in the application of patient-specific iPSCs for disease modeling, drug screening and cell replacement therapy, and discuss their impact on precision personalized medicine.
    Full-text · Article · Jan 2012
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