No abstract

Incubation of murine epidermal cells with delta-aminolevulinic acid (ALA) resulted in a dose- and time-dependent accumulation of porphyrins, predominantly of protoporphyrin. Porphyrin accumulation decreased in the presence of iron, and the iron-mediated decrease was partially reversed by CaMg EDTA (1.25–10.0 mM), suggesting that there is functionally active ferrochelatase in these cells. This study suggests that these cells may be a useful model for the study of cutaneous porphyrin metabolism involving ferrochelatase activity.

T cells are produced by the differentiation of hematopoietic stem and progenitor cells (HSPCs) into Lin− CD34+ CD7+ progenitors that migrate from the bone marrow (BM) to the thymus where further T cell development takes place. The differentiation of HSPCs into T cells can be supported in vitro by using OP9 stromal cells that express a Notch ligand. We have developed a more simple stroma-free culture method that promotes the expansion and differentiation of T cell progenitors from purified CD34+ HSPCs and purified multipotent lymphoid progenitors (MLPs) from cord blood (CB) or BM. CD34+ CB cells isolated using EasySep magnetic separation or CD34+ CD38− CD90− CD45RA+ MLPs further purified by cell sorting were seeded into culture plates coated with an attachment substrate containing Notch ligand and cultured in serum-free StemSpan SFEM II medium in the presence of a cytokine supplement containing SCF, TPO, Flt3L and IL-7. After 3 weeks, CD7+ CD5+ proT cells were detected at a frequency of 84% (range: 63–97%, n=26) with a proT cell yield of 2,130 (142–5,970, n=26) per initial CD34+ cell and 28,110 (4,720–63,930, n=4) per MLP initially seeded. In these cultures 28% (12–43%, n=12) of CD7+ cells also expressed CD1a, indicating further differentiation into preT cells. The pro and preT cells could mature into CD4+ CD8+ double positive T cells (38%; 24–64%, n=6) and TCRαβ+ CD3+ T cells (12%; 5–23%) over two weeks of continued culture in the same medium and in the presence of OP9-DL1 stromal cells. These results demonstrate that HSPCs can expand and differentiate into pro and preT cells under stroma-free and serum-free culture conditions. This novel culture system may benefit research and development of immunotherapies where large numbers of T cells are required.

CD11b+ cells are distributed throughout the body and act as sentinels for the immune system. The frequency of CD11b+ myeloid cells varies in different organs, ranging from approximately 48 % of the brain myeloid (microglia) cells, 30% of bone marrow and 6% of total nucleated cells in gut and spleen. The integrin CD11b heterodimerizes with CD18 to form the mature complex Mac-1. Mac-1 facilitates a variety of immune cell responses including phagocytosis, adhesion, migration and chemotaxis, all functions that are crucial for normal tissue homeostasis and inflammatory responses. CD11b+ cells are difficult and time consuming to isolate, due to their variable frequency using traditional methods. Here, we describe EasySepTM, a simple, column-free immunomagnetic method for the positive selection of highly purified CD11b+ cells from brain, bone marrow, spleen and gut. Labelled CD11b+ cells are retained in a tube in an EasySepTM magnet while unwanted cells are poured off. Selection can be fully automated using RoboSepTM. Starting with a frequency of 7.6 ± 1.3 % CD11b+ cells in spleen, purities of 93.9 ± 4.5 % (n=41) were achieved. Fully competent cells, as shown by functional assays (phagocytosis, ROS), were isolated from the above tissues. This EasySepTM technology provides a fast and simple method to access highly purified functional CD11b+ cells from a variety of tissues, facilitating their study ex-vivo.

Macrophages play an important role in defense against pathogens and in tissue homeostasis. They are classified into two main groups depending on how they are activated: 1) M1 (activated by IFN-γ and LPS) and 2) M2, which include M2a (activated by IL-4 or IL-13), M2b (immune complexes with IL-1β or LPS) and M2c (IL-10) subsets. It is difficult to control macrophage activation and polarization in cultures containing serum, in which variable amounts of M1- and M2-specific cytokines and other factors may be present. Here we describe a serum-free culture system that selectively supports the differentiation of monocytes into either M1 or M2a macrophages. Monocytes were isolated using EasySep immunomagnetic cell separation and cultured for 4 or 6 days in serum-free ImmunoCult medium with 50 ng/mL M-CSF. The cells were then stimulated by adding 10 ng/mL LPS plus 50 ng/mL IFN-γ for M1 or 10 ng/mL IL-4 for M2a macrophage polarization and cultured for 2 more days. The yields of M1 and M2a macrophage were 51±18% and 58±14% (mean±SD), respectively. M1 macrophages expressed high levels of CD80 and CCR7 (96±3% and 62±15%, n=57), but very low levels of CD206 and CD209. In contrast, M2a macrophages expressed CD206 and CD209 (90±10% and 92±13%, n=59), but were negative or low for CD80 and CCR7. M1 macrophages produced TNF-α and IL-12 (mean±SEM: 2821±396 and 656±86, pg/mL, n=24). M2a macrophages produced small amounts of IL-10 (29±6 pg/mL, n=21). Both M1 and M2a macrophages were functional as demonstrated in a phagocytosis assay using fluorescently-labeled E.coli. By selecting appropriate stimuli this culture method can be easily adapted to generate other macrophage subsets as well and should prove useful for the study of macrophage biology.