In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34+ EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34+ EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34+ EB cells, NK and T cell potentials were detected in a CD45− subset, whereas CD45+ EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34+CD45− EB cells showed that CD45+Mac-1−Ter119− ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45−CD34+ EB cells have lymphoid potential, and they differentiate into more mature CD45+Lin− hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122− and they rapidly acquire CD122 as they differentiate along the NK lineage.
Dendritic cells (DCs) control immune responses through their robust antigen presenting activity. Steady-state mouse spleen contains three major DC subsets with distinct functional and phenotypic properties including CD8+ and CD8− conventional DCs (cDCs), and plasmocytoid DCs (pDCs). These subsets are present at a very low frequency (< 4%). Typically, elaborate purification protocols such as FACS-based cell sorting or expansion in culture are needed to obtain enough DCs for subsequent studies. Here, we describe a negative selection method to isolate all DC subsets (panDC) from mouse spleen. This method uses immunomagnetic, column-free cell separation technology (EasySepTM). Briefly, non-DCs are labeled for depletion with biotinylated antibodies and cross-linked to magnetic particles using bispecific antibody complexes. The unwanted cells are then removed using an EasySepTM magnet. The selection steps can be fully automated using RoboSepTM . The panDCs are assessed by flow cytometry and defined as Lin−CD11c+ (cDCs) or Lin−CD11cloPDCA-1+ (pDCs). PanDC purities of 80 ± 7% (n=8) are achieved. The rare pDCs are enriched 36-fold with purity of 11.4 ± 1.4% as compared to 0.3 ± 0.1% in the start spleen. Both CD8+ and CD8− cDC subsets are represented in the cDC fraction. The freshly isolated DCs are not activated but upregulate maturation markers upon stimulation with LPS. This method enables fast, easy isolation of panDCs required for immune regulation studies.
Dendritic cells (DCs) are central regulators of adaptive immune responses that potentially have major applications in the immunotherapy of cancer and autoimmune disorders. DCs are rare in peripheral blood (PB) and are therefore often generated in vitro from PB monocytes. We developed an animal component-free (ACF) and serum-free medium, ImmunoCult-ACF DC, that supports the generation of DCs from monocytes in culture. CD14+ monocytes were isolated using EasySep immunomagnetic separation and cultured for 5 days in ImmunoCult-ACF DC medium and cytokines (GM-CSF and IL-4) to promote their differentiation into CD14−CD83− immature DCs. The immature DCs were then stimulated for 2 days with the ImmunoCult-DC Maturation Supplement, a combination of cytokines and pro-inflammatory mediators, to promote their maturation to CD14− CD83+ DCs (93 ± 5% CD83+, n=23). The yield of mature DCs was 42 ± 23% (n=23), similar to the yield in a control serum-free medium (49 ± 24%, n=16). Mature DCs produced high levels of IL-12, on average 371 pg/mL (range: 27–1756, n=9 in cultures initiated with 106 monocytes). Mature DCs loaded with CMV, EBV and Flu virus peptides efficiently stimulated the proliferation of autologous CD8+ T cells in 7 day co-cultures. An 18-fold increase in T cell numbers (range: 5–31, n=4) was observed when compared to control culture conditions containing only T cells without mature DCs. In conclusion, functional DCs can be efficiently generated by differentiation of monocytes in a completely animal (including human) component-free and serum-free medium. This medium and culture method will enable further research into the development and application of DCs for cellular therapy.
Natural Killer (NK) cells play an important role in innate immunity by secreting proinflammatory cytokines and killing tumor cells and virus-infected cells. Human NK cells can be generated by culturing CD34+ hematopoietic stem and progenitor cells (HSPCs) with stromal cells and cytokines. The use of stroma (and serum) is, however, not desirable in clinical applications. We developed a culture method for generating large numbers of NK cells from purified CD34+ cord blood (CB) HSPCs in the absence of serum and stromal cells. CD34+ CB cells were isolated by EasySep magnetic separation, seeded into culture plates coated with a Notch ligand and cultured for two weeks in StemSpan Serum-Free Expansion Medium (SFEM II) supplemented with SCF, TPO, Flt3L and IL-7. These conditions promote expansion of HSPCs and their differentiation into CD7+CD5+ lymphoid progenitors. Cells were then transferred to non-coated plates and cultured for two more weeks in StemSpan SFEM II supplemented with SCF, Flt3L, IL-7, IL-15 and a small molecule, UM729, to promote differentiation into NK cells. After culture, on average 76% (range: 54–90%, n=12) of cells expressed the NK cell marker CD56 and 58% (range: 25–80%) coexpressed CD56 and NKp46 (a NK cell activating receptor). A more mature CD56+CD16+ NK cell subset also emerged in these cultures (5%, range: 1–11%). The yield of CD56+ NK cells was ~13,000 (600–41,000) per initial CD34+ cell. The NK cells were functional and able to kill K562 target cells. These results show that HSPCs can expand and differentiate into NK cells under stroma- and serum-free culture conditions. This novel culture system will be useful for studies directed toward the development of cancer immunotherapies where large numbers of NK cells are needed.
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