The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the AML1 gene on chromosome 21 and the ETO gene on ...chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human MRP8 promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.
Studies of natural killer (NK) cell function in vivo have been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a ...transgenic mouse with defective natural killing and selective deficiency in NK1.1+CD3-cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acute in vivo rejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1+CD3-cells were responsible for acute tumor rejection, establishing the relationship of NK1.1+CD3-cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNγ in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.
A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly ...their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, ≈ 5% of LT-HSC were in S/G2/M phases of the cell cycle and another 20% were in G1phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription-PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although ≈ 75% of LT-HSC are quiescent in G0at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.
Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic ...human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)+, 5E12+, CD34-, CD45-, and CD24-/lo. Single CD133+CD34-CD45-sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133+CD34-CD45-cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.
The primary role of cytokines in haemato-lymphopoiesis is thought to be
the regulation of cell growth and survival. But the instructive
action of cytokines in haematopoiesis has not been well ...addressed.
Here we show that a clonogenic common lymphoid progenitor,
a bone marrow-resident cell that gives rise exclusively to lymphocytes (T,
B and natural killer cells), can be redirected to the myeloid lineage by stimulation
through exogenously expressed interleukin (IL)-2 and GM-CSF (granulocyte/macrophage
colony-stimulating factor) receptors. Analysis of mutants of the β-chain
of the IL-2 receptor revealed that the granulocyte- and monocyte-differentiation
signals are triggered by different cytoplasmic domains, showing that the signalling
pathway(s) responsible for these unique developmental outcomes are separable.
Finally, we show that the endogenous myelo-monocytic cytokine receptors for
GM-CSF and macrophage colony-stimulating factor (M-CSF) are expressed at low
to moderate levels on the more primitive haematopoietic stem cells, are absent
on common lymphoid progenitors, and are upregulated after myeloid lineage
induction by IL-2. We conclude that cytokine signalling can regulate cell-fate
decisions and propose that a critical step in lymphoid commitment is downregulation
of cytokine receptors that drive myeloid cell development.
It has been proposed that there are at least 2 classes of dendritic cells (DCs), CD8α+ DCs derived from the lymphoid lineage and CD8α− DCs derived from the myeloid lineage. Here, the abilities of ...lymphoid- and myeloid-restricted progenitors to generate DCs are compared, and their overall contributions to the DC compartment are evaluated. It has previously been shown that primitive myeloid-committed progenitors (common myeloid progenitors CMPs) are efficient precursors of both CD8α+ and CD8α− DCs in vivo. Here it is shown that the earliest lymphoid-committed progenitors (common lymphoid progenitors CLPs) and CMPs and their progeny granulocyte-macrophage progenitors (GMPs) can give rise to functional DCs in vitro and in vivo. CLPs are more efficient in generating DCs than their T-lineage descendants, the early thymocyte progenitors and pro-T cells, and CMPs are more efficient DC precursors than the descendant GMPs, whereas pro-B cells and megakaryocyte-erythrocyte progenitors are incapable of generating DCs. Thus, DC developmental potential is preserved during T- but not B-lymphoid differentiation from CLP and during granulocyte-macrophage but not megakaryocyte-erythrocyte development from CMP. In vivo reconstitution experiments show that CLPs and CMPs can reconstitute CD8α+ and CD8α− DCs with similar efficiency on a per cell basis. However, CMPs are 10-fold more numerous than CLPs, suggesting that at steady state, CLPs provide only a minority of splenic DCs and approximately half the DCs in thymus, whereas most DCs, including CD8α+ and CD8α− subtypes, are of myeloid origin.
Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of ...cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.
The aging of tissue-specific stem cell and progenitor cell compartments is believed to be central to the decline of tissue and organ integrity and function in the elderly. Here, we examine evidence ...linking stem cell dysfunction to the pathophysiological conditions accompanying aging, focusing on the mechanisms underlying stem cell decline and their contribution to disease pathogenesis.
Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of ...mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.
Many types of adult tissues, especially for high turnover tissues such as the blood and intestinal system, stand on a hierarchical tissue-specific stem cell system. Tissue-specific stem cells ...concurrently have self-renewal capacity and potential to give rise to all types of mature cells in their tissue. The differentiation process of the tissue-specific stem cell is successive restriction of these capacities. The first progeny of tissue-specific stem cells are multipotent progenitors (MPPs) that lose long-term self-renewal capacity yet have full lineage potential. MPPs in turn give rise to oligopotent progenitors, which then commit into lineage-restricted progenitors. This hierarchical system enables a lifelong supply of matured functional cells that generally have a short life span and a relatively high turnover rate. In this chapter, we review our findings and other key experiments that have led to the establishment of the current cellular stem and progenitor hierarchy in the blood-forming systems of mice and humans for both normal and leukemic hematopoiesis. We also review select signaling pathways intrinsic to normal hematopoietic and leukemic stem cell populations as well our recent findings elucidating the possible origin of the leukemia stem cell.
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