Cytomegalovirus Inhibits the Engraftment of Donor Bone Marrow Cells by Downregulation of Hemopoietin Gene Expression in Recipient Stroma

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.     

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.Subject terms: Haematopoietic stem cells, Stem-cell research     

Latent murine cytomegalovirus DNA in splenic stromal cells of mice.

Latency is an integral feature of the pathogenesis of cytomegalovirus infection and disease. Using in situ hybridization, we detected viral DNA in the splenic stroma of mice with acute murine cytomegalovirus (MCMV) infection but could not detect latent infection. By using enzymatic amplification of a 700-bp region of exon 4 of immediate-early gene 1 of MCMV, viral DNA was consistently detected in whole spleens of latently infected mice. MCMV DNA was detected in 16 of 23 stromal cell fractions from latently infected animals, in only 2 of 13 residual nonstromal cell fractions, and in none of 9 additional lymphocyte or macrophage-enriched nonstromal cell preparations. We conclude that MCMV DNA is maintained predominantly, and possibly exclusively, in stromal cells in the spleens of latently infected mice.     

Ly49H+ NK cells migrate to and protect splenic white pulp stroma from murine cytomegalovirus infection

In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients.     

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 ([Formula: see text]-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.     

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.     

Wild-type measles virus interferes with short-term engraftment of human CD34+ hematopoietic progenitor cells

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.     

Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment

The identity of cells that establish the hematopoietic microenvironment (HME) in human bone marrow (BM), and of clonogenic skeletal progenitors found in BM stroma, has long remained elusive. We show that MCAM/CD146-expressing, subendothelial cells in human BM stroma are capable of transferring, upon transplantation, the HME to heterotopic sites, coincident with the establishment of identical subendothelial cells within a miniature bone organ. Establishment of subendothelial stromal cells in developing heterotopic BM in vivo occurs via specific, dynamic interactions with developing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the HSC "niche" involved in vascular remodeling). Our data reveal the functional relationships between establishment of the HME in vivo, establishment of skeletal progenitors in BM sinusoids, and angiogenesis.     

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.     

Expression of CD23 by human bone marrow stromal cells.

CD23 is a surface antigen expressed by a variety of human hematopoietic cells and shown to display multiple biological functions. In present work, we assayed CD23 expression by human bone marrow (BM) or by stromal cells derived from this tissue. While freshly isolated BM-cells showed low CD23 expression, a subset of long term BM-culture (LTBMC)-derived stromal cells expressed CD23 mRNA at high levels in their steady state and secreted soluble CD23 in their culture supernatants. To assay the role of CD23 in LTBMC, these cultures were initiated in the presence of neutralizing anti-CD23 mAb. A dramatic decrease in total numbers of hematopoietic cells and CFU-GM recovery was observed in these cultures as compared to controls. These data suggest a role of CD23 expression in stroma cell functions and further confirm the ability of this antigen to regulate human hematopoietic cell development.     

Vascular cell adhesion molecule-1 expression and hematopoietic supportive capacity of immortalized murine stromal cell lines derived from fetal liver and adult bone marrow

Ontogeny-specific differences in hematopoietic behavior may be influenced by unique adhesive interactions between hematopoietic cells and the microenvironment, such as that mediated by vascular cell adhesion molecule-1 (VCAM-1, CD 106). Although VCAM-1 is variably expressed during vertebrate development, we hypothesized that VCAM-1 expression might be linked to the enhanced capacity of the fetal liver microenvironment to support hematopoiesis. To test this we used immortalized murine stromal cell lines derived from midgestation fetal liver and adult bone marrow to compare the functional expression of VCAM-1. Molecular analysis of VCAM-1 expression was performed on stromal cell lines using Northern blot analysis, immunoprecipitation studies, and solid-phase enzyme-linked immunosorbent assay. Hematopoietic studies were performed by coculturing fetal liver cells with stromal cell lines, and the functional readout was determined by high-proliferative potential colony-forming cell (HPP-CFC) adherence assays. In contrast to our initial hypothesis, we observed greater expression of VCAM-1 messenger ribonucleic acid and protein on an adult marrow stromal cell line. In functional studies, anti-VCAM-1 antibody inhibited the binding of nearly half of the HPP-CFCs to adult marrow stroma but had a minimal effect on their binding to fetal liver stroma, despite the greater adherence of HPP-CFCs to fetal stroma. We conclude that VCAM-1 influences the hematopoietic supportive capacity of immortalized murine stroma derived from adult bone marrow. Our studies suggest that cellular interactions other than those mediated by VCAM-1 are involved in the increased adhesive capacity of immortalized murine stroma derived from fetal liver.     

Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice

Deficient thymopoiesis is a pivotal determinant of impaired immune competence following hematopoietic stem cell transplantation (HSCT). Stem cell factor (SCF) is essentially involved in early thymopoiesis. We evaluated whether SCF administration would improve recovery of thymopoiesis following HSCT in immunodeficient mice receiving: 1) bone marrow (BM) transplantation of congenic mice; or 2) human fetal liver HSCT in the human immune system mouse model. Following murine BM transplantation, SCF significantly enhanced thymopoiesis and peripheral T cell recovery in lymph nodes and spleen. SCF did not affect BM lymphoid progenitor recovery and/or expansion. Median thymic cellularity increased from 0.9 in PBS- to 266 × 10(4)/thymus in SCF-treated mice (p = 0.05). Following human HSCT in human immune system mice, higher thymic cellularity was observed in SCF-treated mice. Double-negative and early double-positive thymocyte subsets increased, but especially late double-positive, CD4 single-positive, and CD8 single-positive thymocyte subsets were significantly enhanced (p < 0.05). These results show that exogenous supply of SCF may significantly improve murine and human posttransplant thymopoiesis, for which the effect is probably exerted by directly promoting T cell development intrathymically rather than by enhanced entry of prethymically expanded lymphoid progenitors.     

Rescue of myeloid lineage-committed preprogenitor cells from cytomegalovirus-infected bone marrow stroma.

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis.     

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.     

Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the "quiescent" and "proliferative" niches in which hematopoietic stem cells and progenitors reside.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

Efficient Lymphoreticular Prion Propagation Requires PrPc in Stromal and Hematopoietic Cells

In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrP(c)). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrP(c) (Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrP(c) on hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation of Prnp in either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrP(c) expression by both stromal and hematopoietic compartments.