Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγc null-humanized mouse model. The UL133-UL138 NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations.

Summary Human cytomegalovirus (HCMV) continues to be a significant cause of morbidity and mortality in organ transplant recipients despite the availability of antiviral therapy. Considerable controversy exists regarding the use of granulocyte-colony stimulating factor (G-CSF) mobilized blood products from HCMV seropositive donors during stem cell transplantation (SCT) and in patients receiving granulocyte transfusions to treat neutropenia. In order to understand mechanisms of HCMV transmission to patients receiving G-CSF mobilized blood products, we generated a novel NOD-scid IL2Rγcnull humanized mouse model in which HCMV establishes a latent infection in human hematopoietic lineage cells. In this model, G-CSF induces the reactivation of latent HCMV in monocytes/macrophages that have migrated into organ tissues. These results suggest that the use of G-CSF mobilized blood products from seropositive donors pose an elevated risk for HCMV transmission to recipients.

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2 ؊/؊ mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit ؉ Sca-1 ؉ Lineage ؊ (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2 ؊/؊ KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2 ؊/؊ mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2 ؊/؊ KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2 ؊/؊ bone marrow cells. We conclude that Fancd2 ؊/؊ mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies. IntroductionFanconi anemia (FA) is a rare, autosomal, recessive genetic disorder associated with severe birth defects, cancer predisposition, and bone marrow failure. Thirteen causative genes (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, FANCI, FANCL/PHF9/Pog, FANCJ/BRIP1/BACH1, FANCM/Hef, and FANCN/ PALB2) have been identified and cloned to date, and the encoded proteins are believed to work together in a common DNA damageresponse pathway to maintain genomic integrity and protect the genome from DNA damage induced by cross-linking agents. 1,2 Although deficiency in DNA cross-link repair renders all FA cells susceptible to cross-linking agents, bone marrow is the most affected organ system. Mutations in any of the different FA genes almost universally lead to bone marrow failure, which is the primary cause of mortality in FA. 3 The pathogenesis of bone marrow failure in FA remains elusive. Mutations in several genes involved in DNA damage repair, including Atr, XPD, and Ercc1, caused either hematopoietic stem cell (HSC) loss or impaired HSC function under conditions of stress. [4][5][6] These studies suggest that the maintenance of genome integrity is critical for HSC survival and function. However, the extent to which genotoxicity, resulting from impaired DNA damage repair, contributes to bone marrow failure in FA is unclear. 7 Other pathways associated with hematopoietic failure, such as altered cytokine signaling, may also contribute to FA pathogenesis. 8,9 For example, levels of proapoptotic cytokines tumor necrosis factor-␣ (TNF-␣) and interferon-␥ (IFN-␥) are elevated in FA lymphocytes, bone marrow cells, and FA patient serum samples. 10-12 FA bone marrow cells (at least of the C complementation group) are also hypersensitive to these cytokines and undergo apoptosis when exposed to even low levels of them. [13][14][15] To better understand FA, multiple murine knockout models, includingand Fancl Ϫ/Ϫ m...

Bone morphogenetic protein 4 (BMP4) is required for mesoderm commitment to the hematopoietic lineage during early embryogenesis. However, deletion of BMP4 is early embryonically lethal and its functional role in definitive hematopoiesis is unknown. Consequently, we used a BMP4 hypomorph to investigate the role of BMP4 in regulating hematopoietic stem cell (HSC) function and maintaining steady-state hematopoiesis in the adult. Reporter gene expression shows that Bmp4 is expressed in cells associated with the hematopoietic microenvironment including osteoblasts, endothelial cells, and megakaryocytes. Although resting hematopoiesis is normal in a BMP4-deficient background, the number of c-Kit+, Sca-1+, Lineage- cells is significantly reduced. Serial transplantation studies reveal that BMP4-deficient recipients have a microenvironmental defect that reduces the repopulating activity of wild-type HSCs. This defect is even more pronounced in a parabiosis model that demonstrates a profound reduction in wild-type hematopoietic cells within the bone marrow of BMP4-deficient recipients. Furthermore, wild-type HSCs that successfully engraft into the BMP4-deficient bone marrow show a marked decrease in functional stem cell activity when tested in a competitive repopulation assay. Taken together, these findings indicate BMP4 is a critical component of the hematopoietic microenvironment that regulates both HSC number and function.

BackgroundAlthough the lymphatic system arises as an extension of venous vessels in the embryo, little is known about the role of circulating progenitors in the maintenance or development of lymphatic endothelium. Here, we investigated whether hematopoietic stem cells (HSCs) have the potential to give rise to lymphatic endothelial cells (LEC).Methodology/Principal FindingsFollowing the transfer of marked HSCs into irradiated recipients, donor-derived LEC that co-express the lymphatic endothelial markers Lyve-1 and VEGFR-3 were identified in several tissues. HSC-derived LEC persisted for more than 12 months and contributed to ∼3–4% of lymphatic vessels. Donor-derived LECs were not detected in mice transplanted with common myeloid progenitors and granulocyte/macrophage progenitors, suggesting that myeloid lineage commitment is not a requisite step in HSC contribution to lymphatic endothelium. Analysis of parabiotic mice revealed direct evidence for the existence of functional, circulating lymphatic progenitors in the absence of acute injury. Furthermore, the transplantation of HSCs into ApcMin/+ mice resulted in the incorporation of donor-derived LEC into the lymphatic vessels of spontaneously arising intestinal tumors.Conclusions/SignificanceOur results indicate that HSCs can contribute to normal and tumor associated lymphatic endothelium. These findings suggest that the modification of HSCs may be a novel approach for targeting tumor metastasis and attenuating diseases of the lymphatic system.