AC133+ G0 cells from cord blood show a high incidence of long-term culture-initiating cells and a capacity for more than 100 million-fold amplification of colony-forming cells in vitro.

AC133+ cells may provide an alternative to CD34+ cells as a target for cell expansion and gene therapy protocols. We examined the differences in proliferative potential between cord blood selected for AC133 or CD34 in serum-free, stroma cell-free culture for up to 30 weeks. Because most hemopoietic stem cells reside within the G0/G1 phase of the cell cycle, we combined enrichment according to AC133 or CD34 expression with G0 position in the cell cycle to identify populations enriched for putative stem cells. Our results show that AC133+ G0 cells demonstrated a long-term culture-initiating cell incidence of 1 in 4.2 cells, had a colony-forming cell incidence of 1 in 2.8 cells, were capable of producing 660 million-fold expansion of nucleated cells and 120 million-fold expansion of colony-forming units-granulocyte-macrophage over a period of 30 weeks, and were consistently superior to CD34+ G0 cells according to these parameters. Furthermore, we have shown that AC133+CD34- cells have the ability to generate CD34+ cells in culture, which suggests that at least some AC133+ cells are ancestral to CD34+ cells. We conclude that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34 and that, in combination with isolation according to G0 phase of the cell cycle, AC133 isolation identifies a highly enriched population of putative stem cells.

Globin gene activation during haemopoiesis is driven by protein complexes nucleated by GATA-1 and GATA-2.

How does an emerging transcriptional programme regulate individual genes as stem cells undergo lineage commitment, differentiation and maturation? To answer this, we have analysed the dynamic protein/DNA interactions across 130 kb of chromatin containing the mouse alpha-globin cluster in cells representing all stages of differentiation from stem cells to mature erythroblasts. The alpha-gene cluster appears to be inert in pluripotent cells, but priming of expression begins in multipotent haemopoietic progenitors via GATA-2. In committed erythroid progenitors, GATA-2 is replaced by GATA-1 and binding is extended to additional sites including the alpha-globin promoters. Both GATA-1 and GATA-2 nucleate the binding of various protein complexes including SCL/LMO2/E2A/Ldb-1 and NF-E2. Changes in protein/DNA binding are accompanied by sequential alterations in long-range histone acetylation and methylation. The recruitment of polymerase II, which ultimately leads to a rapid increase in alpha-globin transcription, occurs late in maturation. These studies provide detailed evidence for the more general hypothesis that commitment and differentiation are primarily driven by the sequential appearance of key transcriptional factors, which bind chromatin at specific, high-affinity sites.

Molecular signatures of self-renewal, differentiation, and lineage choice in multipotential hemopoietic progenitor cells in vitro.

The molecular mechanisms governing self-renewal, differentiation, and lineage specification remain unknown. Transcriptional profiling is likely to provide insight into these processes but, as yet, has been confined to "static" molecular profiles of stem and progenitors cells. We now provide a comprehensive, statistically robust, and "dynamic" analysis of multipotent hemopoietic progenitor cells undergoing self-renewal in response to interleukin-3 (IL-3) and multilineage differentiation in response to lineage-affiliated cytokines. Cells undergoing IL-3-dependent proliferative self-renewal displayed striking complexity, including expression of genes associated with different lineage programs, suggesting a highly responsive compartment poised to rapidly execute intrinsically or extrinsically initiated cell fate decisions. A remarkable general feature of early differentiation was a resolution of complexity through the downregulation of gene expression. Although effector genes characteristic of mature cells were upregulated late, coincident with morphological changes, lineage-specific changes in gene expression were observed prior to this, identifying genes which may provide early harbingers of unilineage commitment. Of particular interest were genes that displayed differential behavior irrespective of the lineage elaborated, many of which were rapidly downregulated within 4 to 8 h after exposure to a differentiation cue. These are likely to include genes important in self-renewal, the maintenance of multipotentiality, or the negative regulation of differentiation per se.

The human granulocyte/macrophage colony-stimulating factor receptor alpha2 isoform influences haemopoietic lineage commitment and divergence.

A number of alternatively spliced isoforms of haemopoietic growth factor receptors (HGFRs) have been described, but their role in human haemopoiesis remains undetermined. We have investigated the relative expression of the alpha1 and alpha2 isoforms of human granulocyte/macrophage colony-stimulating factor receptor (hGM-CSFR) during haemopoietic cell differentiation, and have shown that both subunits are independently regulated during differentiation of CD34+ human haemopoietic progenitor cells. To further investigate these ex-vivo observations, we established a series of murine FDCP mix cell lines, which, as a consequence of the ectopic expression of alpha1 or alpha2 hGM-CSFR, demonstrated differential differentiation responses to hGM-CSF. In this model system, hGM-CSFR-alpha2-expressing cells showed increased hGM-CSF-mediated erythroid/megakaryocytic differentiation compared with hGM-CSFR-alpha1-expressing cells.

Transcription factor-mediated lineage switching reveals plasticity in primary committed progenitor cells.

The developmental plasticity of transplanted adult stem cells challenges the notion that tissue-restricted stem cells have stringently limited lineage potential and prompts a re-evaluation of the stability of lineage commitment. Transformed cell systems are inappropriate for such studies, since transformation potentially dysregulates the processes governing lineage commitment. We have therefore assessed the stability of normal lineage commitment in primary adult haematopoietic cells. For these studies we have used prospectively isolated primary bipotent progenitors, which normally display only neutrophil and monocyte differentiation in vitro. In response to ectopic transcription factor expression, these neutrophil/monocyte progenitors were reprogrammed to take on erythroid, eosinophil and basophil-like cell fates, with the resultant colonies resembling the mixed lineage colonies normally generated by multipotential progenitors. Clone-marking and daughter cell experiments identified lineage switching rather than differential cell selection as the mechanism of altered lineage output. These results demonstrate that the cell type-specific programming of apparently committed primary progenitors is not irrevocably fixed, but may be radically re-specified in response to a single transcriptional regulator.