Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells

Abstract

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated

Characterization of Dendritic Cells and Macrophages Generated by Directed Differentiation from Mouse Induced Pluripotent Stem Cells

“…The morphological changes observed during the differentiation of the iPS cells into DCs were very similar to that observed in the differentiation culture for ESDCs [2]. However, there was some delay (1-2 days in step 1 and 2 and 2-4 days in step 3) in the kinetics of differentiation process of iPS cells, as in comparison with most of mouse ES cell lines.…”

“…The staining of cells and analysis on a flow cytometer (FACScan, Becton Dickinson, San Jose, CA; http://www.bd.com) was done as described previously [2]. The following monoclonal antibodies (mAb) conjugated with fluorescence isothiocyanate (FITC) or PE were used for staining: anti-mouse Flk-1 (clone Avas12a1, rat IgG2a, eBioscience, San Diego, CA; http://www.ebioscience.com), anti-mouse CD45 (clone 30-F11, rat IgG2b, eBioscience), antimouse CD11b (clone M1/70, IgG2b, Pharmingen), anti-mouse CD11c (clone N148, hamster IgG, Chemicon, Temecula, CA, http://www.chemicon.com), anti-mouse CD80 (clone RMMP-1, rat IgG2a, Caltag), anti-mouse CD86 (clone RMMP-2, rat IgG2a, Caltag), anti-F4/80 (A3-1, rat IgG2b, Serotec Ltd., Oxford, U.K., http://www.serotec.com), mouse IgG2a control (clone G155-178, Pharmingen), mouse IgG2a control (clone G155-178, Pharmingen), rat IgG2a control (clone LO-DNP-16, Caltag), rat IgG2a control (clone LODNP-57, Beckman-Coulter, Fullerton, CA, http://www.…”

“…38C-2 was developed by introduction of the four genes (Oct3/4, Sox2, Klf4, and c-Myc) for reprogramming factors into embryonic fibroblasts and subsequent selection based on the expression of the Nanog gene. The procedure to induce the differentiation of iPS cells into DCs, composed of three steps as shown in supporting information Figure 1, was basically the same as that developed previously using mouse ES cells [2].…”

“…Several groups have previously established methods to generate APC or DCs from mouse [1,2] and human [3][4][5] embryonic stem (ES) cells (ES-DCs). Genetic engineering of ES-DCs can readily be done by the introduction of transgenes into undifferentiated ES cells and subsequent differentiation of the ES cell clones into ES-DCs.…”

Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway

“…Recently, functional dendritic cells were generated from mouse ES cells using the embryoid body method or by coculture of ES cells with a mouse M-CSF-deficient bone-marrow stromal cell line, OP9 (19,20). The OP9 cells efficiently induce multilineage hemopoietic differentiation of mouse as well as nonhuman primate ES cells (21,22).…”