Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs

Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy

Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC.Xenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent.CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

Phosphatidylinositol-3-kinase activation mediates proline-rich tyrosine kinase 2 phosphorylation and recruitment to beta1-integrins in human CD34+ cells

OBJECTIVE: beta1-integrins mediate hematopoietic stem and progenitor cell homing and retention in the bone marrow (BM) and inhibit hematopoietic proliferation and differentiation. Having no intrinsic kinase activity, integrins recruit intracellular kinases, such as the focal adhesion kinase (FAK) or the related proline-rich tyrosine kinase 2 (PYK2), to initiate signal transduction. Phosphatidylinositol-3-kinase (PI3K), which is involved in beta1-integrin signaling in many cell types, is physically and functionally associated with FAK in anchorage-dependent cells. Because PYK2 is the principal focal adhesion kinase expressed in primary human CD34+ cells, we assessed its functional relationship with PI3K in CD34+ cells in response to integrin engagement. METHODS: beta1-integrins on primary mobilized peripheral blood CD34+ cells and CD34+ KG1A cells were engaged by adhesion to fibronectin (FN) or by cross-linking with an anti-beta1 integrin antibody, respectively. PI3K activity and PYK2 phosphorylation were then assessed in the presence or absence of the PI3K inhibitor, wortmannin. Association between PI3K, PYK2, and the beta1-integrin subunit were also evaluated in co-immunoprecipitation experiments. RESULTS: beta1-integrin engagement induced PI3K activation, which was required for, and temporally preceded, PYK2 phosphorylation, indicating that PI3K lies upstream of PYK2 in CD34+ cells. Furthermore, although PYK2 and PI3K were constitutively associated, interaction of the PYK2/PI3K complex with beta1-integrins required prior integrin engagement and PI3K activation. CONCLUSION: Activation of PI3K following beta1-integrin engagement on human CD34+ cells results in subsequent phosphorylation of PYK2, and is required for the recruitment of the PI3K/PYK2 complex to beta1-integrins at the cell surface.status: publishe

Integrin engagement-induced inhibition of human myelopoiesis is mediated by proline-rich tyrosine kinase 2 gene products

OBJECTIVE: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells. METHODS: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays. RESULTS: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors. CONCLUSION: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.status: publishe