Generation of Functional Neutrophils from a Mouse Model of X-Linked Chronic Granulomatous Disorder Using Induced Pluripotent Stem Cells

et al. (2011) Generation of Functional Neutrophils from a Mouse Model of X-Linked Chronic Granulomatous Disorder Using Induced Pluripotent Stem Cells. PLoS ONE 6(3): e17565. doi:10.1371/journal.pone.0017565 Generation of Functional Neutrophils from a Mouse Model of X-Linked Chronic Granulomatous Disorder Using Induced Pluripotent Stem Cells Sayandip Mukherjee 0 Giorgia Santilli 0 Michael P. Blundell 0 Susana Navarro 0 Juan A. Bueren 0 Adrian J. 0 Georg Hacker, University Freiburg, Germany 0 1 Centre for Immunodeficiency, UCL Institute of Child Health , London , United Kingdom , 2 Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energe ticas, Medioambientales y Tecnolo gicas (CIEMAT) , Madrid , Spain , 3 Great Ormond Street Hospital NHS Trust , London , United Kingdom Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91phox is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils. - Funding: This work was supported by generous funding from the following: European Program "7FWP, Health" (PERSIST; Ref Grant Agreement no: 222878); Chronic Granulomatous Disorder Research Trust (J4G/04B/GT09); Biotechnology and Biological Sciences Research Council, UK (BB/F015526/1); Wellcome Trust (090233/Z/09/Z); GOSH Childrens Charity; Ministry of Science and Innovation for Programa de Fomento de Cooperacio n Cientfica Internacional (110-90.1); Plan Nacional de Salud y Farmacia (SAF 2009-07164); Fondo de Investigaciones Sanitarias, ISCIII (Programa RETICS-RD06/0010/0015); Fundacio n Marcelino Botn for promoting translational research at the Divisio n de Hematopoyesis y Terapia Genica at the CIEMAT-CIBERER. CIBERER is an initiative of the Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The successful application of induced pluripotent stem cell (iPSC) technology in murine models of human diseases, and in generating disease-free autologous cells from patient samples offers considerable potential for development of personalized cell based therapies of monogenic disorders [19]. In this study, we have studied a mouse model of X-linked chronic granulomatous disorder (X-CGD)[10]. CGD is a group of inherited immunodeficiency disorders resulting from mutations in any one of five subunits of the NADPH-oxidase found in neutrophils and other phagocytic leukocytes. Patients with CGD typically present early in life with recurrent and life-threatening infections due to impaired killing of ingested microbes. Two-thirds of patients with CGD have mutations in the X-linked CYBB gene on chromosome Xp21.1 encoding membrane bound gp91phox (where phox stands for phagocyte oxidase). X-CGD in human patients can be cured by haematopoietic stem cell transplantation from HLA genotypicallymatched donors with high rate of success. Gene therapy using gammaretroviral vectors has also proved to be useful for short term treatment of life-threatening infection, although complicated by insertional mutagenesis. Treatment of those patients without HLA-matched donors remains problematic. In addition to haematopoietic stem cell (HSC) based therapy, refractory infections in CGD patients can be successfully treated using repeated infusions of functional allogeneic neutrophils, although this strategy often results in exaggerated inflammation and alloimmunisation[1114]. In this study, we provide a proof-ofprinciple that iPSC technology can provide a valuable platform for investigating gene therapeutic approaches in CGD. Results and Discussion Induced pluripotent stem cells (iPSCs) generated from adult fibroblasts of X-CGD mice were adapted to feeder-free condition for five passages and subsequently characterized for stem cell morphology (round shape, large nucleus, and scant cytoplasm), alkaline phosphatase activity, and expression of pluripotency markers Sox2, Oct4, Klf4, Nanog, SSEA-1, and c-Myc (Fig. 1A). Based on these results, a single clone was selected for future experiments henceforth designated as cgd-iPSC. As control, an iPSC clone from wild-type mice of identical background was also obtained which will be referred to as wt-iPSC. The ability to generate teratoma in immunodeficient mice constitutes an important test of pluripotency. Sub-cutaneous injection of cgdiPSC into immunodeficient mice generated tumour between the 4th and 5th week and subsequent histological analysis of tumour sections revealed the presence of ectodermal (neural tube), mesodermal (cartilage) and endodermal (gut epithelium) structures as shown in figure 1B. Immunostaining revealed the presence of definitive markers for all three germinal layers in these sections (Figure 1C) thereby confirming the tumour growth as a teratoma. Complete silencing of retroviral transgenes marks the attainment of a fully reprogrammed pluripotent state. This is immensely critical for the employment of iPSC in multi-lineage differentiation protocols[15,16]. As shown in figure 1D, we could not detect any expression of the exogenous reprogramming factors from the retroviral vectors in cgd-IPSC (passage five) when compared to transduced fibroblasts (day three). Expressions of endogenous reprogramming factor transcripts were consistently detected in cgd-IPSC when cultured and propagated in embryonic stem cell media. Our next goal was to differentiate the cgd-iPSC line to cells of myeloid lineage. Myeloid differentiation is a complex process involving coordinated binding of haematopoietic cytokines (notably granulocyte-colony stimulating factor and IL-6) to their cognate receptors in a stage and lineage specific manner resulting in the derivation of mature granulocytes or monocytes/macrophages. The scheme for differentiation is outlined in figure 2A and is adapted from previously published protocols for in-vitro haematopoietic differentiation of mouse embryonic stem cells and iPSC with minor modifications[17]. RNA expression profiling of six day old embryoid bodies (EBs) showed down regulation of expression of the pluripotency markers (Nanog and Oct4), with concomitant upregulation in expression of differentiation markers including Nestin (ectoderm), a-fetoprotein (...truncated)