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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.01.036 Byline: Derrick J. Rossi (1), Catriona H.M. Jamieson (2), Irving L. Weissman (3) Abstract: 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. Author Affiliation: (1) Immune Disease Institute, Harvard Stem Cell Institute, and the Department of Pathology, Harvard Medical School, Boston, MA 02115, USA (2) Moores Cancer Center, Division of Hematology/Oncology and Department of Medicine, University of California, San Diego, CA 92093, USA (3) Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Cancer Center, and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5323, USA

Stem cell lineages are legitimate units of natural selection and in a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. Data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.

The AP-1 transcription factor JunB regulates the numbers of hematopoietic stem cells (HSC). JunB overexpression decreases the frequency of long-term HSC, while JunB inactivation specifically expands the number of long term HSC and granulocyte/macrophage progenitors resulting in chronic myeloproliferative disorder (MPD).

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.04.006 Byline: Derrick J. Rossi (1), Irving L. Weissman (1)(2) Abstract: Self-renewal pathways crucial for maintaining stem cells are deregulated in cancer, raising the spectre that cancer therapies targeting such pathways might also ablate normal stem cells. As report in a recent Nature paper, this may not be the case for the tumor suppressor protein Pten, which drives the self-renewal of normal hematopoietic stem cells and the formation of leukemia cells through different mechanisms. Author Affiliation: (1) Department of Pathology, Stanford University School of Medicine, Beckman Center B259, 279 Campus Drive, Stanford, CA 94305, USA (2) Institute of Stem Cell Biology and Regenerative Medicine and Developmental Biology, Stanford University School of Medicine, Beckman Center B259, 279 Campus Drive, Stanford, CA 94305, USA

Phenotypic, morphological, and functional criteria are used to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. The clonal isolation of functional adult myogenic progenitors is described, demonstrating that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+ HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/- Rag2-/- II2rg-/- mouse model of liver failure. [ABSTRACT FROM AUTHOR]

Summary The contribution of the different waves and sites of developmental hematopoiesis to fetal and adult blood production remains unclear. Here, we identify lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) as a marker of yolk sac (YS) endothelium and definitive hematopoietic stem and progenitor cells (HSPCs). Endothelium in mid-gestation YS and vitelline vessels, but not the dorsal aorta and placenta, were labeled by Lyve1-Cre . Most YS HSPCs and erythro-myeloid progenitors were Lyve1-Cre lineage traced, but primitive erythroid cells were not, suggesting that they represent distinct lineages. Fetal liver (FL) and adult HSPCs showed 35%–40% Lyve1-Cre marking. Analysis of circulation-deficient Ncx1 −/− concepti identified the YS as a major source of Lyve1-Cre labeled HSPCs. FL proerythroblast marking was extensive at embryonic day (E) 11.5–13.5, but decreased to hematopoietic stem cell (HSC) levels by E16.5, suggesting that HSCs from multiple sources became responsible for erythropoiesis. Lyve1-Cre thus marks the divergence between YS primitive and definitive hematopoiesis and provides a tool for targeting YS definitive hematopoiesis and FL colonization. [ABSTRACT FROM AUTHOR]

Summary The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271 + melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2 + /VEGFR1 + ), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271 + melanoma cells represents a powerful therapeutic approach against metastatic melanoma. [ABSTRACT FROM AUTHOR]

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research. [Display omitted] • Inhibiting WNT signaling improves culture of chimp and pig-tailed macaque iPSCs • Endogenous high expression of BCL2 in the rhesus inner cell mass • Expression of BCL2 enhances proliferation of chimp iPSCs in rhesus early embryo • Co-culture of chimp and human iPSCs can differentiate into integrated cardiomyocytes Roodgar et al. demonstrate an improved culture of chimpanzee and pig-tailed macaque iPSCs. They show high expression of endogenous BCL2 in rhesus macaque ICM. Through over expression of BCL2 in chimpanzee iPSCs, they generate primate cross-species pre-implantation embryos composed of chimpanzee iPSCs and rhesus pre-implantation embryos. [ABSTRACT FROM AUTHOR]

Summary: The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life. [Copyright &y& Elsevier]

Hematopoietic progenitor cell replacement therapy remains a surprisingly unrefined process. In general, unmanipulated bone marrow or mobilized peripheral blood (MPB) grafts which carry potentially harmful passenger cells are administered after treating recipients with high-dose chemotherapy and/or radiotherapy to eradicate malignant disease, eliminate immunologic barriers to allogeneic cell engraftment, and to ‘make space’ for rare donor stem cells within the stem cell niche. The sequalae of such treatments are substantial, including direct organ toxicity and nonspecific inflammation that contribute to the development of graft-versus-host disease (GVHD) and poor immune reconstitution. Passenger tumor cells that contaminate autologous hematopoietic grafts may contribute to relapse post-transplant. Use of antibodies to rid grafts of unwanted cell populations, and to eliminate or minimize the need for nonspecifically cytotoxic therapies used to condition transplant recipients, will dramatically improve the safety profile of allogeneic and gene-modified autologous hematopoietic stem cell therapies. [ABSTRACT FROM AUTHOR]

Multiple lines of investigation have demonstrated that the immune system plays an important role in preventing tumor initiation and controlling tumor growth. Accordingly, many cancers have evolved diverse mechanisms to evade such monitoring. While multiple immune cell types mediate tumor surveillance, recent evidence demonstrates that macrophages, and other phagocytic cells, play a key role in regulating tumor growth through phagocytic clearance. In this review we highlight the role of tumor immune evasion through the inhibition of phagocytosis, specifically through the CD47–signal-regulatory protein-α pathway, and discuss how targeting this pathway might lead to more effective cancer immunotherapies. [Copyright &y& Elsevier]

Objective: Mouse embryonic stem cells have demonstrated potential to restore infarcted myocardium after acute myocardial infarction. Although the underlying mechanism remains controversial, magnetic resonance imaging has provided reliable in vivo assessment of functional recovery after cellular transplants. Multimodal comparison of the restorative effects of mouse embryonic stem cells and mouse embryonic fibroblasts was performed to validate magnetic resonance imaging data and provide mechanistic insight. Methods: SCID-beige mice (n = 55) underwent coronary artery ligation followed by injection of 2.5 × 105 mouse embryonic stem cells, 2.5 × 105 mouse embryonic fibroblasts, or normal saline solution. In vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells was evaluated by (1) in vivo pressure–volume loops, (2) in vivo bioluminescence imaging, and (3) ex vivo TaqMan (Roche Molecular Diagnostics, Pleasanton, Calif) polymerase chain reaction and immunohistologic examination. Results: In vivo magnetic resonance imaging demonstrated significant improvement in left ventricular ejection fraction at 1 week in the mouse embryonic stem cell group. This finding was validated with (1) pressure–volume loop analysis demonstrating significantly improved systolic and diastolic functions, (2) bioluminescence imaging and polymerase chain reaction showing superior posttransplant survival of mouse embryonic stem cells, (3) immunohistologic identification of cardiac phenotype within engrafted mouse embryonic stem cells, and (4) polymerase chain reaction measuring increased expressions of angiogenic and antiapoptotic genes and decreased expressions of antifibrotic genes. Conclusion: This study validates in vivo magnetic resonance imaging as an effective means of evaluating the restorative potential of mouse embryonic stem cells. [Copyright &y& Elsevier]

Abstract: Dr. Irving Weissman was the honored E. Donnall Thomas lecturer at the Tandem BMT Meetings, held on February 10, 2007, at Keystone, Colorado. Dr. Weissman has been a major player, and has provided us with enormous insight into many areas of biology, dating back to his high school days in Montana. He led an enormously productive career at Stanford University where he has taught us many lessons involving our understanding of lymphocyte homing, stem cell biology, both of the hematopoietic system and other types of stem cells, and also now, about cancer stem cells. Dr. Weissman has made enormous contributions to this burgeoning field that has provided us new insights and new opportunities for treatment strategies. In addition to a very productive laboratory career, he is also currently the director of both the Stem Cell Institute, as well as the Cancer Center at Stanford University. The following text is a modified transcribed version of the presentation made by Dr. Weissman. [Copyright &y& Elsevier]

Cancer stem cells (CSCs) are cells that drive tumorigenesis, as well as giving rise to a large population of differentiated progeny that make up the bulk of the tumor, but that lack tumorigenic potential. CSCs have been identified in a variety of human tumors, as assayed by their ability to initiate tumor growth in immunocompromised mice. Further characterization studies have demonstrated that gene expression profiles in breast cancer correlate with patient prognosis, and brain CSCs are specifically resistant to radiation through DNA damage repair. In addition, specific signaling pathways play a functional role in CSC self renewal and/or differentiation, and early studies indicate that CSCs are associated with a microenvironmental niche. Thus the biological properties of CSCs are just beginning to be revealed, and the continuation of these studies should lead to the development of CSC-targeted therapies for cancer treatment. [Copyright &y& Elsevier]

Summary: Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event. [Copyright &y& Elsevier]

Colonial organisms provide a unique experimental system for stem cell biology. The colonial Urochordate Botryllus schlosseri reproduces sexually as well as by continuous asexual budding. Adjacent colonies with a shared histocompatibility allele undergo vascular fusion and establish a common blood circulation, performing natural transplantation. Fused colonies become chimeras, often with complete somatic replacement of the host cell genotype by the fused parabiont. We attempted to establish a radioprotection assay for the somatic stem cells that induce long-term chimerism in Botryllus. We demonstrate over a range of radiation doses that neither autologous nor allogeneic cell transplantation enhances survival of host colonies. This suggests that high mitotic index associated with continuous asexual development leads to radiosensitivity of organs and structures essential to survival during engraftment. We observe that radiation induces uncontrolled epithelial cell proliferation in abnormally terminated buds, suggesting that stem cells are not required for the initial stages of bud development. [Copyright &y& Elsevier]

Colonial tunicates are the only chordate that possess two distinct developmental pathways to produce an adult body: either sexually through embryogenesis or asexually through a stem cell-mediated renewal termed blastogenesis. Using the colonial tunicate Botryllus schlosseri , we combine transcriptomics and microscopy to build an atlas of the molecular and morphological signatures at each developmental stage for both pathways. The general molecular profiles of these processes are largely distinct. However, the relative timing of organogenesis and ordering of tissue-specific gene expression are conserved. By comparing the developmental pathways of B. schlosseri with other chordates, we identify hundreds of putative transcription factors with conserved temporal expression. Our findings demonstrate that convergent morphology need not imply convergent molecular mechanisms but that it showcases the importance that tissue-specific stem cells and transcription factors play in producing the same mature body through different pathways. • Embryogenesis and blastogenesis have different morphological and molecular programs • Distinct molecular programs can lead to the same outcome • Tissue-specific transcriptional timings are shared in both developmental pathways • The expression and timing of many transcription factors are conserved in development Kowarsky et al. present a developmental atlas of colonial tunicates. They show how sexual and asexual reproduction processes, despite having largely distinct molecular programs, share similar timing of organ formation. Tissue-specific stem cells and transcription factors display similar genetic dynamics in different developmental pathways to produce the same organism. [ABSTRACT FROM AUTHOR]

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases. • CD47 "don't-eat-me" signals are upregulated on infected cells • Antibody-mediated blockade of CD47 enhances APC function in vivo • Therapeutic CD47 blockade enhances T cell responses and speeds LCMV clearance • CD47 blockade works in a pathogen non-specific manner to enhance immunity Cham et al. describe a way to enhance natural immune responses to infections by blocking interactions between two molecules (CD47 and SIRPα) that normally put brakes on the immune system. Since this therapy targets the immune system, it could have broad applicability against a wide range of infectious agents. [ABSTRACT FROM AUTHOR]

Leukemia stem cells are defined as transformed hematopoietic stem cells or committed progenitor cells that have amplified or acquired the stem cell capacity for self-renewal, albeit in a poorly regulated fashion. In this issue of Cancer Cell, Huntly and colleagues report a striking difference in the ability of two leukemia-associated fusion proteins, MOZ-TIF2 and BCR-ABL, to transform myeloid progenitor populations. This rigorous study supports the idea of a hierarchy among leukemia-associated protooncogenes for their ability to endow committed myeloid progenitors with the self-renewal capacity driving leukemic stem cell propagation, and sheds new light on the pathogenesis of chronic and acute myelogenous leukemias. [Copyright &y& Elsevier]

In this issue of Cancer Cell, Goardon et al. revise earlier conclusions regarding acute myelogenous leukemia (AML) stem cells by demonstrating that in the majority of patients, they reside in two hierarchically related populations most similar to normal hematopoietic progenitors. These findings have implications for therapeutic targeting of these cells. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are defined by their ability to (i) fully recapitulate the tumor of origin when transplanted into immunodeficient mouse hosts, and (ii) self-renew, demonstrated by their ability to be serially transplanted. These properties suggest that CSCs are required for tumor maintenance and metastasis; thus, it has been predicted that CSC elimination is required for cure. This prediction has profoundly altered paradigms for cancer research, compelling investigators to prospectively isolate CSCs to characterize the molecular pathways regulating their behavior. Many potential strategies for CSC-directed therapy have been proposed, but few studies have rigorously demonstrated their efficacy using in vivo models. Herein, we highlight recent studies that demonstrate the utility of CSC-directed therapies and discuss the implications of the CSC hypothesis to experimental design and therapeutic strategies.Molecular Therapy (2009) 17 2, 219–230 doi:10.1038/mt.2008.254 [ABSTRACT FROM AUTHOR]

Abstract: Advancing age is frequented by the onset of a variety of hematological conditions characterized by diminished homeostatic control of blood cell production. The fact that upstream hematopoietic stem and progenitor cells are obligate mediators of homeostatic control of all blood lineages, has implicated the involvement of these cells in the pathophysiology of these conditions. Indeed, evidence from our group and others has suggested that two of the most clinically significant age-associated hematological conditions, namely, the diminution of the adaptive immune system and the elevated incidence of myeloproliferative diseases, have their origin in cell autonomous changes in the functional capacity of hematopoietic stem cells. [Copyright &y& Elsevier]

: ObjectiveAlthough migration of hematopoietic stem cells (HSC) is essential for normal hematopoiesis and successful hematopoietic cell transplantation, little is known about the mechanisms that underlie this movement. We have sought to characterize the factors that regulate HSC migration by analyzing changes in expression of particular adhesion receptors associated with cyclophosphamide/granulocyte colony-stimulating factor (Cy/G-CSF)–induced HSC mobilization.: MethodsExpression by Lineage−/loThy1.1loSca-1+c-kit+ HSC of members of the β1 integrin family of adhesion molecules was assessed in untreated or Cy/G-CSF–treated mice by multiparameter flow cytometry. In parallel, the in vivo homing properties of normal and mobilized HSC were compared following intravenous transfer of fluorescently marked HSC.: ResultsNormal adult HSC express high levels of several β1 integrin family members. Following Cy/G treatment, bone marrow HSC selectively downregulate α2 integrin expression and upregulate α5 expression. HSC found in the blood following Cy/G-CSF treatment express significantly lower levels of multiple integrins than their bone marrow and/or splenic counterparts. Changes in integrin expression by blood-borne HSC correlate with a 50% decrease in their ability to home to the bone marrow in short-term assays, and with previously observed defects in competitive engraftment by these HSC. Similar reductions in bone marrow (BM) homing are observed for BM HSC treated with α4 integrin function blocking mAb prior to injection. Modulation of integrin expression induced by mobilization was not associated with cell-cycle progression.: ConclusionChanges in integrin expression and function are associated with HSC mobilization and likely significantly affect the engraftment potential of hematopoietic stem cells. [Copyright &y& Elsevier]

Purpose: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR.Methods and Materials: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed.Results: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors.Conclusions: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR. [ABSTRACT FROM AUTHOR]

• Botryllus schlosseri HSCs have been isolated and their niche identified. • Mammalian and B. schlosseri HSCs and their respective niches share many homologous transcripts. • Cytotoxicity is the mechanism of immune allogeneic cellular rejection. • Botryllus Histocompatibility Factor (BHF) determines fusion or rejection of colonies. • Allogeneic rejection by cytotoxic cells is inhibited by BHF, similar to NK cells. Evolution and selection have shaped diverse immune systems throughout phylogeny, the vast majority of which remain unexplored. Botryllus schlosseri is a colonial tunicate, a sister group to vertebrates, that develops as a chordate, then metamorphoses to an asexually reproductive invertebrate that every week makes the same body plan from budded stem cells. Genetically distinct B. schlosseri colonies can fuse to form a chimera, or reject each other based on allogeneic recognition. In chimeras, circulating germline and somatic stem cells participate in development; stem cells compete in all individuals in the fused colonies, with rejection preventing germline parasitism. Here we review the isolation and characterization of B. schlosseri hematopoietic stem cells (HSC) and their niches, and the role of the immune effector cells in allorecognition. [ABSTRACT FROM AUTHOR]

Nanomedicine offers unique advantages in treating human cancers. However, physiological and pathological barriers within normal and disease tissues, which are highly variable among individuals, often hinder its effectiveness. The body possesses specific innate responses to nanoparticles (NPs), which when combined with unique pathophysiological signatures in the tumor microenvironment, can severely limit the utility of nanomedicine in the oncological setting. Furthermore, with the successes of cancer immunotherapies, understanding nanoimmune interactions and developing immune-smart cancer nanomedicine that can take advantage of the body's immune functions will increasingly become clinically relevant. Therefore, a better understanding of the important native and acquired biological processes that dictate the fate of nanomedicine is integral to developing more effective individualized platforms for treating cancer patients. [ABSTRACT FROM AUTHOR]

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage- mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a "don't eat me signal" that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8+ T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a "cytokine storm". Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity. [ABSTRACT FROM AUTHOR]

Summary: The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify “cell islands” (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life. [ABSTRACT FROM AUTHOR]

Metastatic breast cancer remains a major treatment challenge. The use of high-dose chemotherapy (HDCT) with rescue by autologous mobilized peripheral blood (MPB) is controversial, in part because of contamination of MPB by circulating tumor cells. CD34+Thy-1+ selected hematopoietic stem cells (HSC) represent a graft source with a greater than 250,000-fold reduction in cancer cells. Here, we present the long-term outcome of a pilot study to determine feasibility and engraftment using HDCT and purified HSC in patients with metastatic breast cancer. Twenty-two patients who had been treated with standard chemotherapy were enrolled into a phase I/II trial between December 1996 and February 1998, and underwent HDCT followed by rescue with CD34+Thy-1+ HSC isolated from autologous MPB. More than 12 years after the end of the study, 23% (5 of 22) of HSC recipients are alive, and 18% (4 of 22) are free of recurrence with normal hematopoietic function. Median progression-free survival (PFS) was 16 months, and median overall survival (OS) was 60 months. Retrospective comparison with 74 patients transplanted between February 1995 and June 1999 with the identical HDCT regimen but rescue with unmanipulated MPB indicated that 9% of patients are alive, and 7% are without disease. Median PFS was 10 months, and median OS was 28 months. In conclusion, cancer-depleted HSC following HDCT resulted in better than expected 12- to 14-year PFS and OS in a cohort of metastatic breast cancer patients. These data prompt us to look once again at purified HSC transplantation in a protocol powered to test for efficacy in advanced-stage breast cancer patients. [ABSTRACT FROM AUTHOR]

Tumor immunosurveillance is a well-established mechanism for regulation of tumor growth. In this regard, most studies have focused on the role of T- and NK-cells as the critical immune effector cells. However, macrophages play a major role in the recognition and clearance of foreign, aged, and damaged cells. Macrophage phagocytosis is negatively regulated via the receptor SIRPα upon binding to CD47, a ubiquitously expressed protein. We recently showed that CD47 is up-regulated in myeloid leukemia and migrating hematopoietic progenitors, and that the level of protein expression correlates with the ability to evade phagocytosis. These results implicate macrophages in the immunosurveillance of hematopoietic cells and leukemias. The ability of macrophages to phagocytose tumor cells might be exploited therapeutically by blocking the CD47-SIRPα interaction. [Copyright &y& Elsevier]

Objective: The aim of this study was to determine the lineage origin of interferon-α–producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs. Materials and Methods: Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-α production after virus challenge. Results: Transplantation of 1 × 104 common myeloid progenitors, 1 × 104 common lymphoid progenitors or 2.5 × 104 granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology. Conclusion: Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin. [Copyright &y& Elsevier]

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes. [Copyright &y& Elsevier]

We demonstrate here that “promiscuous” expression of myeloid or lymphoid genes precedes lineage commitment in hematopoiesis. Prospectively purified single common myeloid progenitors (CMPs) coexpress myelo-erythroid but not lymphoid genes, whereas single common lymphoid progenitors (CLPs) coexpress T and B lymphoid but not myeloid genes. Genes unrelated to the adopted lineage are downregulated in bipotent and monopotent descendants of CMPs and CLPs. Promiscuous gene expression does not alter the biological potential of multipotent progenitors: CMPs with an activated endogenous M lysozyme locus yield normal proportions of myelo-erythroid colonies, and CLPs expressing the pre-T cell receptor α gene differentiate into normal numbers of B cells. Thus, the accessibility for multiple myeloid or lymphoid programs promiscuously may allow flexibility in fate commitments at these multipotent stages. [Copyright &y& Elsevier]

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit donor HSC engraftment to provide life-long hematopoietic and immune function. Currently HCT relies on DNA damaging radiation or chemotherapy to achieve HSC niche clearance. We have pursued a non-toxic approach to target and deplete HSC using humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit). We opened a Phase 1 dose escalation trial using AMG 191 as the sole conditioning agent to achieve donor HSC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID). The primary endpoint is to assess the safety of administering AMG 191. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor granulocyte chimerism at 24 weeks. We have completed the first dose cohort of patients receiving 0.1 mg/kg AMG 191 and treated the first two patients in the second cohort (0.3 mg/kg). All five patients tolerated the AMG 191 infusion and the subsequent infusion of their CD34-selectd donor cells without clinical problems. Here we report efficacy in the first two patients, who have reached the 24-week timepoint. Both patients had T-B-NK+ SCID with mutations in the DCLRE1C (Artemis) gene. Both previously received unconditioned HCT as infants, failed to develop donor B cells and remained dependent on exogenous immunoglobulin. CD34-selected mobilized peripheral blood cells from the original donors were infused when the AMG 191 serum level was <100 ng/mL. Beginning at 8 weeks post-HCT both patients showed evidence of increasing peripheral blood CD19+CD20+ B cells. Extended immunophenotype analysis demonstrated that naïve B cells were present, and chimerism studies confirmed that the B cells were of donor origin. The first patient had impaired T cell function prior to his HCT. Significant increases in both naïve CD4+ and CD8+ T cells were observed with clinical improvement beginning at 22 weeks post-HCT. Importantly, starting at 8 weeks these 2 patients demonstrated donor chimerism in purified granulocytes, an indicator of HSC engraftment, which rose from 0% pre-HCT to ≥5% at 24 weeks (Table 1). Conclusion: These data are proof of concept that a humanized monoclonal antibody targeting CD117 can safely clear human HSC niches and facilitate donor HSC engraftment. This study is ongoing and open for enrollment. [ABSTRACT FROM AUTHOR]