Heather E. Fleming

Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.

Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche

Stem cells reside in a specialized, regulatory environment termed the niche that dictates how they generate, maintain and repair tissues. We have previously documented that transplanted haematopoietic stem and progenitor cell populations localize to subdomains of bone-marrow microvessels where the chemokine CXCL12 is particularly abundant. Using a combination of high-resolution confocal microscopy and two-photon video imaging of individual haematopoietic cells in the calvarium bone marrow of living mice over time, we examine the relationship of haematopoietic stem and progenitor cells to blood vessels, osteoblasts and endosteal surface as they home and engraft in irradiated and c-Kit-receptor-deficient recipient mice. Osteoblasts were enmeshed in microvessels and relative positioning of stem/progenitor cells within this complex tissue was nonrandom and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different haematopoietic cell subsets localized to distinct locations according to the stage of differentiation. When physiological challenges drove either engraftment or expansion, bone-marrow stem/progenitor cells assumed positions in close proximity to bone and osteoblasts. Our analysis permits observing in real time, at a single cell level, processes that previously have been studied only by their long-term outcome at the organismal level.

Wnt Signaling in the Niche Enforces Hematopoietic Stem Cell Quiescence and Is Necessary to Preserve Self-Renewal In Vivo

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells.

Cytokine Signaling and Hematopoietic Homeostasis Are Disrupted in Lnk-deficient Mice

The adaptor protein Lnk, and the closely related proteins APS and SH2B, form a subfamily of SH2 domain-containing proteins implicated in growth factor, cytokine, and immunoreceptor signaling. To elucidate the physiological function of Lnk, we derived Lnk-deficient mice. Lnk −/− mice are viable, but display marked changes in the hematopoietic compartment, including splenomegaly and abnormal lymphoid and myeloid homeostasis. The in vitro proliferative capacity and absolute numbers of hematopoietic progenitors from Lnk − / − mice are greatly increased, in part due to hypersensitivity to several cytokines. Moreover, an increased synergy between stem cell factor and either interleukin (IL)-3 or IL-7 was observed in Lnk − / − cells. Furthermore, Lnk inactivation causes abnormal modulation of IL-3 and stem cell factor–mediated signaling pathways. Consistent with these results, we also show that Lnk is highly expressed in multipotent cells and committed precursors in the erythroid, megakaryocyte, and myeloid lineages. These data implicate Lnk as playing an important role in hematopoiesis and in the regulation of growth factor and cytokine receptor–mediated signaling.

Hematopoietic Stem Cell Responsiveness to Exogenous Signals Is Limited by Caspase-3

Limited responsiveness to inflammatory cytokines is a feature of adult hematopoietic stem cells and contributes to the relative quiescence and durability of the stem cell population in vivo. Here we report that the executioner Caspase, Caspase-3, unexpectedly participates in that process. Mice deficient in Caspase-3 had increased numbers of immunophenotypic long-term repopulating stem cells in association with multiple functional changes, most prominently cell cycling. Though these changes were cell autonomous, they reflected altered activation by exogenous signals. Caspase-3(-/-) cells exhibited cell type-specific changes in phosphorylated members of the Ras-Raf-MEK-ERK pathway in response to specific cytokines, while notably, members of other pathways, such as pSTAT3, pSTAT5, pAKT, pp38 MAPK, pSmad2, and pSmad3, were unaffected. Caspase-3 contributes to stem cell quiescence, dampening specific signaling events and thereby cell responsiveness to microenvironmental stimuli.

Mechanisms of selection mediated by interleukin-7, the preBCR, and hemokinin-1 during B-cell development

Summary:  Many of the stromal‐derived signals and factors that regulate B lymphopoiesis have been identified. We review recent evidence from our laboratory that shows that there are at least three phases during B‐cell development when cells direct their own maturation, independent of stromal cells. Following the expression of the preB‐cell receptor (preBCR), cells acquire the ability to proliferate in low levels of interleukin‐7 (IL‐7), which acts as a self‐selecting mechanism to expand cells that have successfully expressed a preBCR in environments that are non‐permissive to preBCR– cells. Second, the preBCR is required for a contact‐mediated event between B‐cell progenitors. Disruption at this stage prevents the further maturation of progenitors to the lipopolysaccharide (LPS)‐responsive stage. Finally, the transition from IL‐7 receptor to mature antigen receptor‐based signaling is enhanced by a novel member of the tachykinin family, hemokinin‐1. This series of maturation, survival, and differentiation signals is generated by B‐lineage cells as they progress through developmental checkpoints on the way to becoming functionally mature cells.

Frontline: IL‐7 does not prevent pro‐B/pre‐B cell maturation to the immature/sIgM+ stage

IL‐7 plays many fundamental roles during murine B lineage development. One reported function is to maintain progenitors in a developmentally immature state by preventing differentiation to the surface IgM (sIgM)+ stage. Withdrawal of IL‐7 from cultures has been shown to lead to increases in mature traits such as RAG expression, IgL rearrangements and expression of sIgM. These observations have been interpreted as an inductive event promoting the differentiation of progenitor cells. In contrast to this, we reproducibly observe sIgM+ cells that have differentiated in cultures containing IL‐7. We find that sIgM+ cells arise as a normal consequence when B lineage cells are cultured in the presence of IL‐7. However, these cells are short‐lived and are quickly replaced by newly emerging sIgM+ cells that differentiate from sIgM– progenitors. Withdrawal of IL‐7 from these cultures only prevents the survival and proliferation of CD2–sIgM– pro‐B cells but does not change the number of cells that differentiate to the sIgM+ stage. This changes the ratio of sIgM–:sIgM+ cells and results only in an apparent maturation of the culture as a whole. Therefore withdrawal of IL‐7 from these cultures acts as a selection event, not an induction event, for populations that are normally present.

Embryonic stem cells make human T cells

Bit by bit the barriers to achieving differentiation of desired somatic cells from human embryonic stem cells (hESC) are falling away. In a recent issue of PNAS, Galic et al. (1) reported that hESC can be made to differentiate to mature T cells. Prior reports have shown the capacity of mouse ESC (mESC) to accomplish this process, and getting hESC to form other blood elements has been well described. To some, it would seem then that yet another report of hESC becoming a cell type of interest is no big deal. The cells are after all pluripotent and should make any and all mature cell types. The challenge, of course, is to actually drive the cells down a particular lineage pathway and ultimately to be able to do this on command. Getting there requires some serious spade work: first, showing that hESC lines can become the cell of interest; second, developing a system to accomplish it with high frequency and purity; and third, using that system to define the specific molecular cues that are necessary and sufficient. The field is still in need of the first step, and those who work through it deserve much credit.