Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

Human embryonic stem cells (hESCs) randomly differentiate into multiple cell types during embryoid body (EB) development. To date, characterization of specific factors capable of influencing hematopoietic cell fate from hESCs remains elusive. Here, we report that the treatment of hESCs during EB development with a combination of cytokines and bone morphogenetic protein-4 (BMP-4), a ventral mesoderm inducer, strongly promotes hematopoietic differentiation. Hematopoietic progenitors of multiple lineages were generated from EBs and were found to be restricted to the population of progeny expressing cell surface CD45. Addition of BMP-4 had no statistically significant effect on hematopoietic differentiation but enabled significant enhancement in progenitor self-renewal, independent of cytokine treatment. Hematopoietic commitment was characterized as the temporal emergence of single CD45 ؉ cells first detectable after day 10 of culture and was accompanied by expression of hematopoietic transcription factors. Despite the removal of cytokines at day 10, hematopoietic differentiation of hESCs continued, suggesting that cytokines act on hematopoietic precursors as opposed to differentiated hematopoietic cells. Our study establishes the first evidence for the role of cytokines and BMP-4 in promoting hematopoietic differentiation of hESC lines and provides an unprecedented system to study early developmental events that govern the initiation of hematopoiesis in the human.

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here, we identify a newly discovered human repopulating cell, distinct from previously identified repopulating cells, that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells, or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR, Thy-1 and CD34). In contrast to CD34+ subfractions, Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations, providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation.