On the subject of acute myeloid leukemia (AML), there is little consensus about the target cell within the hematopoietic stem cell hierarchy that is susceptible to leukemic transformation, or about the mechanism that underlies the phenotypic, genotypic and clinical heterogeneity. Here we demonstrate that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) - termed the SCID leukemia-initiating cell, or SL-IC - possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all subtypes of AML analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34++ CD38-, similar to the cell-surface phenotype of normal SCID-repopulating cells, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation. The SL-ICs were able to differentiate in vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy.

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+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi 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+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy.

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by f luorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic͞severe combined immunodeficient (NOD͞SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34 ؉ CD38؊ cells. The highly purified SRC were capable of extensive proliferation in NOD͞ SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400,000 progeny 6 weeks after the transplant. Detailed f low cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34 ؉ CD38 ؊ cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and͞or expanding these cells during in vitro culture.