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A Robust and Highly Efficient Immune Cell Reprogramming System

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A Robust and Highly Efficient Immune Cell Reprogramming System View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell Stem Cell Resource A Robust and Highly Efficient Immune Cell Reprogramming System Lars H. Bussmann,1,4 Alexis Schubert,1,4 Thien Phong Vu Manh,1,4 Luisa De Andres,1 Sabrina C. Desbordes,1,2 Maribel Parra,1 Timo Zimmermann,1 Francesca Rapino,1 Javier Rodriguez-Ubreva,2 Esteban Ballestar,2 and Thomas Graf1,3,* 1Center for Genomic Regulation and Universitat Pompeu Fabra, 08003 Barcelona, Spain 2Bellvitge Institute for Biomedical Research (IDIBELL), l’Hospitalet de Llobregat, 08907 Barcelona, Spain 3Institucio Catalana de Recerca I Estudis Avancats, Catalonia 4These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.stem.2009.10.004 SUMMARY embryonic stem cells into the trophectoderm by Cdx2 (Niwa et al., 2005). Direct reprogramming of specialized cells has Here we describe a lineage reprogramming system also been shown in vivo. Thus, an activated form of Pdx1 induces consisting of a B cell line with an estradiol-inducible hepatic cells to turn into pancreatic beta islet cells in Xenopus form of C/EBPa where cells can be converted into (Horb et al., 2003). More recently, a combination of Pdx1 with macrophage-like cells at 100% efficiency within 2 to Ngn1 and MafA was shown to reprogram exocrine cells into 3 days. The reprogrammed cells are larger, contain beta islet cells in the mouse pancreas (Zhou et al., 2008). Given altered organelle and cytoskeletal structures, are enough knowledge about how lineages are reprogrammed, it might therefore one day become feasible to directly custom- phagocytic, and exhibit an inflammatory response. make any desired cell type in cultures or in patients. Time-lapse experiments showed that the cells To unravel the molecular basis of lineage reprogramming, a cell acquire a macrophage morphology and increased system would be desirable where reprogramming can be induced migratory activity as early as 10 hr. During induction, with high efficiency and within a short time span and cell numbers thousands of genes become up- or downregulated, are not limiting. Several of these requirements are met by the including several dozen transcription and chro- conversion of primary lymphoid cells into myeloid cells. For matin-remodeling factors. Time-limited exposure of example, the enforced expression of C/EBPa by retroviral infection cells to the inducer showed that the reprogrammed can convert more than 60% committed B and T cell progenitors cells become transgene independent within 1 to 2 into macrophages (Xie et al., 2004; Laiosa et al., 2006b). The days. The reprogramming can be inhibited, at least frequency of induced lymphoid to macrophage conversions is partially, by perturbation experiments with B cell therefore substantially higher compared to the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by embry- and macrophage transcription factors. The tightness, onic stem cell-associated transcription factors (Takahashi and robustness, and speed of the system described make Yamanaka, 2006). However, even primary lymphoid cells are not it a versatile tool to study biochemical and biological ideal because an important fraction of the cells are resistant to re- aspects of lineage reprogramming. programming, the cultures require stromal cells, and cell numbers are limiting. In addition, for every experiment the starting population INTRODUCTION hastobe freshlyisolatedfrom bonemarrow and infected with retro- viruses, and the infected cells have to be sorted before analyses. Harold Weintraub’s laboratory showed that a single gene, en- Here we describe a pre-B cell line that can be converted by coding the transcription factor MyoD, can induce the differentia- C/EBPa into macrophage-like cells at 100% efficiency within 2 to tion of fibroblasts into myotubes (Davis et al., 1987). The subse- 3 days in the absence of stroma. The induced cells exhibit dramatic quent finding that MyoD can also induce the expression of changes in gene expression within hours and acquire functional muscle genes in cell lines of ectodermal and endodermal origin macrophage properties. Time-lapse experiments showed that all (Weintraub et al., 1989) raised the possibility that fully specialized cells change in morphology and become highly motile. Further- cells can be reprogrammed into other cell types. Indeed, exper- more, we showed that the system can be used to test for genes iments with avian myeloid leukemia cells showed that GATA-1 with the capacity to specifically perturb cell reprogramming. can convert the cells into megakaryocytic/erythroid cells. The cells not only activated genes of the megakaryocytic/erythroid RESULTS lineage but also downregulated macrophage genes (Kulessa et al., 1995). Since then, transcription factors have been used Pre-B Cell Lines Containing C/EBPaER Can Be Induced to reprogram a number of other tissues, such as cultured astro- to Reciprocally Regulate Lineage Marker Expression cytes into neuronal cells by Pax6 (Heins et al., 2002); pancreatic and to Change Cell Parameters at 100% Efficiency cells into liver cells by activation of C/EBPb (Shen et al., 2003); To develop a reprogrammable cell line system, we screened two B cells into macrophages by C/EBPa (Xie et al., 2004); and adult and one fetal pre-B cell lines for their ability to convert into 554 Cell Stem Cell 5, 554–566, November 6, 2009 ª2009 Elsevier Inc. Cell Stem Cell Immune Cell Reprogramming Figure 1. Inducible Reciprocal Regulation A 5’5’ LTR LTR C/EBPaERC/EBPα IRESIRES EGFP 3´3’ LTR LTR of Differentiation Markers in the C10 Cell Line Days after induction B (A) C/EBPaER retroviral construct. 0 1 2 3 4 (B) FACS plot of Mac-1 and CD19 expression in C/ EBPaER-infected cells (C10 cell line) at different times after induction with b-estradiol. (C and D) Kinetics of differentiation-specific cell Mac-1 surface antigens of b-estradiol-induced cells and ethanol-treated control C10 cells, showing median CD19 values of three separate experiments with stan- CDE dard deviation. Blue lines, CD19 expression; red 100 Day 0 Day 3 lines, Mac-1. 80 (E) Morphology of the cells before and after induc- 60 tion, via interference contrast microscopy. Scale bar represents 10 mm. 40 (F) FACS plots showing side scatter (a measure of 20 granularity) and forward scatter (a measure of cell % ag. pos. cells 0 size) of the cells shown in (B). 01234 0 1234 Days after induction F Days after induction the cells became heterogeneous in size 0 1 2 3 4 and shape after prolonged culturing (Fig- ures S2B–S2D). To reveal structural changes, C10 cells were induced for 3 days, fixed, stained, Granularity (SSC) and photographed with a fluorescence Cell volume (FSC) microscope. Staining of mannosidase II revealed reorganization of the Golgi apparatus from a symmetric to a polar macrophages after ectopic expression of C/EBPa. To overcome structure located at one side of the nucleus, facing away from the problem that uninfected B cells rapidly overgrow the reprog- the lamellipodium at the leading edge of the cell (Figure 2A). rammed cells, we generated an inducible virus, containing Staining of F-actin revealed massive actin accumulations at the a fusion between C/EBPa and the estrogen hormone binding leading edge, while the trailingedgeshowed the formation ofstress domain (C/EBPaER), as well as GFP, as an infection marker fibers and increased concentration of Mac-1 antigen. By contrast, (Figure 1A). GFP-positive clones isolated from the lines were in uninduced C10 cells, both actin and CD19 antigen were infected with this virus, treated with b-estradiol, and analyzed symmetrically distributed around the cell periphery (Figure 2B). for Mac-1 (CD11b) and CD19 expression at different times there- after. Clones from the fetal-derived cell line HAFTL responded Induced Cells Exhibit Phagocytic Capacity and Respond most completely and rapidly and one clone, C10, was selected to an Inflammatory Stimulus for further studies. HAFTL cells, which contain a normal, diploid Activated macrophages are highly phagocytic and respond to karyotype of 20 chromosomes (Figure S1 available online), incoming bacterial pathogens with cytokine production and resemble pre-B cells (B220+CD19+, CD43ÀIgMÀ), and will be inflammation (Gordon and Taylor, 2005). To test whether the called ‘‘B cells’’ in the following. Treatment of C10 cells with reprogrammed cells acquire these properties, C10 cells were b-estradiol induced a substantial increase in Mac-1 expression induced for 3 days and tested. They indeed show high phagocytic and downregulation of CD19 already within 1 day, and after activity when incubated with fluorescently labeled E. coli, whereas 3 days essentially 100% of the cells showed a reciprocal regula- uninduced cells were essentially negative (Figures 3A–3C). Then, tion of these markers (Figures 1B and 1C). The kinetics of induced induced cells were treated with LPS or kept unstimulated. As antigen expression changes were highly reproducible, with shown by the qRT-PCR results in Figure 3D, they responded values varying less then 10% in repeat experiments. Induced with a 30- to 1000-fold increased expression of RNAs encoding C10 cells showed an increase in size and granularity (Figures TNF-a, IL-6, IL-1b, and Ccl3 (Mip1a). The data also show that 1E and 1F) and most cells became adherent (Figure S2A). No for some of these markers (IL-1b and Ccl3), the induced cells changes in antigen expression or cellular parameters were seen already expressed elevated levels before LPS stimulation. in solvent-treated C10 cells (Figure 1D) or HAFTL cells treated with b-estradiol. We also determined their growth and survival Time-Lapse Experiments Show Rapid Changes properties. Whereas untreated C10 cells kept growing and then in Morphology, Migratory Activity, and Induction slowed down after 3 days because of overcrowding, induced of Membrane Ruffling cells doubled in number within the first day and then remained Next, we performed time-lapse experiments to observe the quiescent.
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