Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

Unrelated cord blood transplantation (CBT) has now become more common, but as yet there have been only a few reports on its outcome compared with bone marrow transplantation (BMT), especially for adults. We studied the clinical outcomes of 113 adult patients with hematologic malignancies who received unrelated BM transplants (n ‫؍‬ 45) or unrelated CB transplants (n ‫؍‬ 68). We analyzed the hematopoietic recovery, rates of graftversus-host disease (GVHD), risks of transplantation-related mortality (TRM) and relapse, and disease-free survival (DFS) using Cox proportional hazards models. The time from donor search to transplantation was significantly shorter among CB transplant recipients (median, 2 months) than BM transplant recipients (median, 11 months; P < .01). Multivariate analysis demonstrated slow neutrophil (P < .01) and platelet (P < .01) recoveries in CBT patients compared with BMT patients. Despite rapid tapering of immunosuppressants after transplantation and infrequent use of steroids to treat severe acute GVHD, there were no GVHD-related deaths among CB transplant recipients compared with 10 deaths of 24 among BM transplant recipients. Unrelated CBT showed better TRM and DFS results compared with BMT (P ‫؍‬ .02 and P < .01, respectively), despite the higher human leukocyte antigen mismatching rate and lower number of infused cells. These data strongly suggest that CBT could be safely and effectively used for adult patients with hematologic malignancies. (Blood. 2004;104:3813-3820)

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases.

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decayaccelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC. Cancer Res; 72(10); 2609-21. Ó2012 AACR.

We have constructed an allosterically controllable novel enzyme (designated maxizyme) that can be transcribed in vivo under the control of a human tRNA(Val) promoter. The maxizyme has sensor arms that can recognize target sequences, and in the presence of such a target sequence only, it can form a cavity that can capture catalytically indispensable Mg2+ ions. As a target for a demonstration of the potential utility of the maxizyme, we chose BCR-ABL mRNA, the translated products of which cause chronic myelogenous leukemia. Only the maxizyme (but not conventional ribozymes) had extremely high specificity and high-level activity, not only in vitro but also in cultured cells including BV173 cells derived from a patient with a Philadelphia chromosome. The maxizyme induced apoptosis only in leukemic cells with this chromosome.

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs. Stem Cells 2005;23:1304-1313 This material is protected by U.S.

The Pebpb2 gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2͞core binding factor (PEBP2͞CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function, Pebpb2 was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proved lethal in embryos around embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver was severely impaired. The observed phenotype was indistinguishable from that reported for homozygous disruption of AML1, which encodes a DNA binding subunit of PEBP2͞CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2͞CBF in vivo and that PEBP2͞CBF plays an essential role in the development of definitive hematopoiesis.

tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob −/− mice than in wildtype mice. tob −/− p53 −/− mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.Supplemental material is available at http://www.genesdev.org. There is accumulating evidence that genes involved in the negative control of cell growth can function as tumor suppressors. In humans, tob, tob2, ana, pc3b, btg1, and btg2 comprise a family (tob family) of antiproliferative genes (Bradbury et al. 1991;Fletcher et al. 1991;Rouault et al. 1992;Matsuda et al. 1996;Guehenneux et al. 1997;Yoshida et al. 1998;Ikematsu et al. 1999;Buanne et al. 2000). Exogenous expression of Tob family proteins suppresses growth of NIH-3T3 cells by inhibiting G1 progression of the cell cycle (Yoshida et al. 1998;Ikematsu et al. 1999;Guardavaccaro et al. 2000;Maekawa et al. 2002;Suzuki et al. 2002). We showed previously that Tob is a substrate of Erk MAPK, and unphosphorylated Tob suppresses cell-cycle entry of quiescent cells. Erk phosphorylation of Tob blocks the antiproliferative activity (Maekawa et al. 2002;Suzuki et al. 2002), which, at least in part, describes the importance of Erk activation in the cells stimulated by growth factors. When Tob is depleted, Cyclin D1 continues to be expressed and readily progress into S phase during serum starvation (Suzuki et al. 2002). In addition, the antiproliferative activity of Tob is impaired in the presence of exogenously coexpressed Cyclin D1 (Suzuki et al. 2002). These data suggest that tob functions as a tumor suppressor. However, possible involvement of Tob in tumorigenesis and roles of Tob in the control of cyclin D1 expression are unclear.Tob family proteins associate with transcription factors. Virtually all of the Tob family members interact with Caf1 (Rouault et al. 1998;Ikematsu et al. 1999;Yoshida et al. 2001), whose yeast homolog is a component of the CCR4-NOT transcriptional complex (Albert et al. 2000). The CCR4-NOT complex participates in the control of specific sets of genes such as those involved in the late mitotic phase of the cell cycle (Liu et al. 1997). Both BTG1 and BTG2 associate with HoxB9 and estrogen receptor ␣, and modulate their transcription activity (Prevot et al. 2000(Prevot et al. , 2001. Tob associates with Smads transcription complex and affects Smad-mediated gene expression (Yoshida et al. 2000;Tzachanis et al. 2001). This suggests that Tob family proteins are regulators of gene transcription, functioning as either coactivators or corepressors.Here, we report that mice lacking tob are prone to spontaneous formation o...