In mouse, a subset of dendritic cells (DCs) known as CD8α⁺ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α⁺ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α⁺ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α⁺ DCs, human DNGR-1⁺ BDCA3^hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α⁺ DCs) TLR9. DNGR-1⁺ BDCA3^hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1⁺ BDCA3⁺ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8⁺ T cells upon treatment with poly I:C. The characterization of human DNGR-1⁺ BDCA3^hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

Immunodeficient mice are increasingly used to assay human hematopoietic repopulating cells as well as leukemia-initiating cells. One method commonly used to isolate these rare cells is to sort cells stained with fluorochrome-conjugated antibodies into fractions, then transplant the different fractions into immunodeficient mice to test their repopulating ability. The antibodies are generally treated as being neutral in terms of their effects on the experiment. Human repopulating cells are thought to express CD34 and lack CD38. Here we present evidence that anti-CD38 antibodies have a profound inhibitory effect on engraftment of cord blood and leukemia cells. We show that this effect is Fc-mediated and can be overcome by treating mice with immunosuppressive antibodies. When this inhibitory effect is prevented, we demonstrate that the CD34+CD38+ fraction of certain acute myeloid leukemia samples contains all, or at least most, leukemia-initiating cell capacity. This study highlights the potential pitfall of antibody-mediated clearance of repopulating cells and is important for any groups working with this model. More importantly, the work suggests that there is greater variation in the phenotypes of leukemia-initiating cells than previously suggested.

Murine mesenchymal stem cells are capable of differentiating in vitro into different lineages under stimulation with certain cytokines, growth factors and chemicals. However, the true capacitiy of these cells to contribute to different cell-types in vivo is still unclear, especially under minimal injury conditions. In this study, we describe a method of purifying murine mesenchymal stem cells from bone marrow and efficiently transducing them using a lentivirus vector expressing the eGFP reporter gene. Lentivirus-transduced mesenchymal stem cells retained their in vitro ability to differentiate into adipocytes, osteocytes and chondrocytes as well as into myocyte- and astrocyte-like cells. eGFP-mesenchymal stem cells were delivered systemically into minimally injured syngeneic mice. Tracking and tissue-specific differentiation were determined by PCR and immunohistochemistry, respectively. We found donor-derived hepatocytes, lung epithelial cells, myofibroblasts, myofibers and renal tubular cells in some of the recipient mice. Our data indicate that even in the absence of substantial injury, phenotypically defined murine mesenchymal stem cells could acquire tissue specific morphology and antigen expression and thus contribute to different tissue cell-types in vivo.

It is believed that a primitive cell type that maintains the mesenchymal compartment exists in the bone marrow. However, this putative mesenchymal stem/progenitor cell is yet to be identified and isolated. We are reporting the identification, isolation, and detailed characterization of the most primitive mesenchymal progenitor cells in the adult murine bone marrow, based on the expression of stage-specific embryonic antigen–1 (SSEA-1). This primitive subset can be identified in mesenchymal cell cultures and also directly in the bone marrow, thus ascertaining for the first time their existence in an adult organism. Characterization of SSEA-1⁺ mesenchymal cells revealed that upon purification these cells gave rise to SSEA-1⁻ mesenchymal cells, whereas the reverse could not be observed. Also, these SSEA-1⁺ cells have a much higher capacity to differentiate than their negative counterparts, not only to several mesenchymal cell types but also to unconventional cell types such as astrocyte-, endothelial-, and hepatocyte-like cells in vitro. Most importantly, a single-cell–derived population was capable of differentiating abundantly into different mesenchymal cell types in vivo. Altogether we are proposing a hierarchical organization of the mesenchymal compartment, placing SSEA-1⁺ cells at the apex of this hierarchy.