• "Silent" Polymorphisms: How Silent Are They? (Cover Story)
• Pinpointing the Endothelial Cell Precursors
• Flossing May Prevent Plaque (of a Different Sort)!
• Novel Mouse Models of Human Myeloma
• Predicting Outcomes in CLL
• Rituximab in ITP – When and Why Does It Work?
• Expansion Hopes Within Reach?
• Enhancing Hematopoietic Function with Parathyroid Hormone

Pinpointing the Endothelial Cell Precursors

Lilli Petruzzelli, MD, PhD

Dr. Petruzzelli indicated no relevant conflicts of interest.

Yoder MC, Mead LE, Prater D, et al. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 2007;109:1801-9.

Endothelial progenitor cells (EPCs) are the focus of intense investigation because of a need to define the cells for their use as potential therapeutic tools in conditions such as cardiovascular damage and to understand their relationship to hematopoietic precursors. Although animal models indicate that new vessel formation following injection of endothelial progenitor cells is a promising therapeutic intervention, the findings are not recapitulated in humans, and the true endothelial potential of EPCs is under question. This paper re-addresses the isolation and definition of the population of cells that form endothelial cells (ECFCs; endothelial colony-forming cells) and compares them to CFU-EC (endothelial colony-forming units) that are defined by commercially available assays. Comparing two distinct cell culture strategies, the investigators established that ECFCs originate from adherent cells whereas CFU-ECs arise from a non-adherent population. Yoder et al. note that CFU-ECs express hematopoietic markers and, more specifically, have myeloid regenerating capacity but no ability to form blood vessels. CFU-ECs differentiate into phagocytic macrophages, whereas the EPCs proliferate and form vessels, but do not form hematopoietic cells. Finally, taking advantage of the newly described JAK-2 mutation, the investigators put forth an argument that because all CFU-ECs but only a small fraction of ECFC contain the mutation, the former are derived from a hematopoietic precursor and the two populations are not clonally related.

The connection between early hematopoietic precursors and the ability to form blood vessels or support blood cell development continues to be defined, and this paper moves us one step closer to understanding the divide between these processes. The investigators do a careful job of delineating differences between the two populations of cells and note that previous identification of the precursor cells was defined by cell surface markers that may have selected for a population of cells with hematopoietic potential. Their assay relies on growth and colony formation in cell culture on defined surfaces and demonstrates that, from a pooled population of mononuclear blood cells, there were cells capable of both replicating and forming blood vessels that did not have hematopoietic markers. In contrast, the CFU-ECs cells did not replicate and were not capable of forming blood vessels. In part, the identification of unique populations may be obscured by the selection markers used, and, as the authors point out, the vascular formation potential of the cells was not reviewed — this may be a necessary component of analysis. With clearer insight into the cells of interest, it may be time to revisit cell-based therapy and characterize the molecular connection between these two populations of cells.

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Flossing May Prevent Plaque (of a Different Sort)!

Roy Silverstein, MD

Dr. Silverstein indicated no relevant conflicts of interest.

Tonetti MS, D'Aiuto F, Nibali L, et al. Treatment of periodontitis and endothelial function. N Engl J Med 2007;356:911-20.

Can aggressive treatment of periodontal disease decrease chronic systemic inflammation and thereby improve vascular function and slow progression of atherosclerosis? To address this intriguing question, investigators from Connecticut and London performed a randomized, blinded clinical study of a group of otherwise healthy subjects with objectively defined severe periodontitis. One group was treated once with a standard oral hygiene regimen, while the others received an aggressive protocol that included extensive plaque scaling, dental extractions, and topical antibiotics. Subjects were then monitored for six months with serial assessments of endothelial function, oral health, and plasma markers of inflammation, coagulation, and endothelial cell "activation." While the control group showed minimal or no change in these markers, on the day after therapy those subjects treated with the intensive regimen demonstrated a remarkable spike in markers of inflammation, including neutrophil count, CRP, and IL-6, with a more modest increase in markers of coagulation and endothelial activation (vWF, PAI-1, and soluble E-selectin). These data are consistent with the systemic entry of periodontal pathogens that is known to occur after therapy of this type. Within a week, levels of the markers began to drop, and by six months they were back to or below baseline. Of greater interest, beginning at about one month, the intensively treated group showed improvement in endothelial function, assessed by ultrasound measurement of flow-mediated dilation (FMD) of the brachial artery. At the end of the study, these subjects had further improvement in FMD with lower neutrophil counts and soluble E-selectin levels than the controls.

An association between chronic inflammation and atherothrombotic disorders is supported by abundant epidemiologic and laboratory evidence. A pathological hallmark of early atherosclerosis is accumulation of inflammatory leukocytes in the vessel wall, and among the best predictors of atherothrombotic risk are circulating levels of "markers" of chronic inflammation (e.g., leukocyte count, CRP, factor VIII, fibrinogen, and myeloperoxidase). Patients with chronic inflammatory diseases such as lupus and rheumatoid arthritis are known to be at increased risk for coronary disease and stroke. Recently, chronic periodontal infection has been hypothesized to represent a potentially reversible source of chronic systemic inflammation, and periodontal pathogens have been associated with activation of inflammatory cells in the atheromatous vessel wall. The potential importance of this hypothesis is supported by the population incidence of severe periodontitis, which is as high as 3 percent in some estimates. Using a simple, non-invasive assessment of endothelial function that tests the integrity of endothelial-dependent vasodilating pathways (mostly nitric oxide), these authors demonstrated that a single intensive treatment of severe periodontal disease was associated with a sustained improvement in dental health, endothelial function, and markers of chronic inflammation. It is important to note that the endpoints of this study were surrogate markers for inflammation and vascular health, and that subjects were monitored for only six months, but nevertheless the data are consistent with known pathophysiological data and support the need for a larger study examining the effects of intensive treatment of periodontal disease on atherosclerosis progression and coronary events.

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Novel Mouse Models of Human Myeloma

Kenneth Anderson, MD

Dr. Anderson indicated no relevant conflicts of interest.

Carrasco DR, Sukhdeo K, Protopopova M, et al. Targeted overexpression of the transcription factor XBP-1 in B cells promotes plasma cell and lymphoplasmacytic neoplasms in transgenic mice. Cancer Cell, in press.

The majority of studies to date of human multiple myeloma (MM) cells in vivo have utilized xenograft models. Specifically, human MM cells have been injected subcutaneously into SCID mice in order to evaluate the ability of agents to inhibit tumor cell growth and associated angiogenesis, as well as prolong host survival^1-3. A model in which fluorochrome-labeled human MM cells are injected via the tail vein into SCID mice and then migrate and grow primarily in bone has allowed for gene microarray and proteomic studies in MM cells vs. BM as MM cells bind in the BM milieu, both before and after novel drug treatments⁴. Direct injection of MM cells into fetal bone chips implanted subcutaneously into SCID mice (SCID hu model) allows for the study of human MM cells in the context of human extracellular matrix proteins and accessory cells in vivo⁵. This model has been refined by injecting fluorochrome-labeled cytokine-dependent MM cells directly into human bone grafts within SCID mice, which allows for evaluation of cellular and gene changes triggered in tumor vs. BM by MM cell binding, both before and after treatment with novel agents^6,7. These models have therefore delineated the genetic changes and sequelae induced when MM cells bind to the BM, and conversely validated the ability of novel agents to abrogate these genetic changes and induce tumor cell cytotoxicity even in the BM milieu.

XBP-1 is a transcription factor required for plasma cell differentiation, which is also highly expressed at a gene and protein level in MM cells vs. normal plasma cells^8-10. Interleukin-6 (IL-6) upregulates both transcript and protein levels of XBP-1 in MM cells; conversely, knockdown of XBP-1 decreases viability and sensitizes MM cells to dexamethasone. The novel agent, 2-methoxyestradiol, downregulates XBP-1 transcripts and protein in MM cells⁹, whereas the proteasome inhibitor Bortezomib works, at least in part, by targeting XBP-1 and the unfolded protein response⁸.

In this paper, Carrasco and colleagues describe a genetically based model of MM, generated by overexpression of XBP-1 spliced isoform (XBP-1s) in the lymphoid system, which faithfully reflects human MM. Specifically, mice transgenic for Eu-directed XBP-1s develop pathognomonic features of monoclonal gammopathy of unclear significance (MGUS), which progress to MM with time, including serum monoclonal protein, bone marrow plasmacytosis, renal disease, and lytic bone disease. In this model, genomic analysis of premalignant B cells and MM cells showed dysregulation of genes with known relevance to human MM, including cyclin D1, MAF, MAFB, and uncovered pathogenetic insights into MCL-1 and FOS/JUN. This model therefore offers a unique opportunity to identify genetic changes mediating the development of MGUS, as well as the progression to MM. Overlay of additional genetic abnormalities (p16, p53) offers the opportunity to shorten the prolonged latency time of this model and define their role in MM pathogenesis. Finally, this model provides a unique system both for identifying novel targets and validating novel targeted therapies.

1. LeBlanc R, Catley L, Hideshima T, et al. Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model. Cancer Res 2002;62:4996-5000.
2. Lentzsch S, Rogers MS, LeBlanc R, et al. S-3-Amino-phthalimido-glutarimide inhibits angiogenesis and growth of B-cell neoplasias in mice. Cancer Res 2002;62:2300-2305.
3. Lentzsch S, LeBlanc R, Podar K, et al. Immunodulatory analogs of thalidomide inhibit growth of HS Sultan cells and angiogenesis in vivo. Leukemia 2003;17:41-44.
4. Mitsiades CS, Mitsiades N, Bronson RT, et al. Fluorescence imaging of multiple myeloma cells in a clinically-relevant SCID/NOD in vivo model: biologic and clinical implications. Cancer Res 2003;63:6689-6696.
5. Urashima M, Chen BP, Chen S, et al. The development of a model for the homing of multiple myeloma cells to human bone marrow. Blood 1997;90:754-765.
6. Tassone P, Neri A, Burger R, et al. A clinically relevant SCID-hu in vivo model of human multiple myeloma. Blood 2005;106:713-716.
7. Tassone P, Neri P, Burger R, et al. Combination therapy with IL-6 receptor super-antagonist Sant7 and dexamethasone induces antitumor effects in a novel SCID-hu in vivo model of human multiple myeloma. Clin Cancer Res 2005;11:4251-4258.
8. Lee AH, Iwakoshi NN, Anderson KC, Glimcher LH. Proteasome inhibitors disrupt the unfolded protein response in myeloma cells. Proc Natl Acad Sci USA 2003;100:9946-9951.
9. Chauhan D, Li G, Hideshima T, et al. Identification of genes regulated by 2-methoxyestradiol in multiple myeloma cells using oligonucleotide arrays. Blood 2003;101:3606-3614.
10. Munshi N, Hideshima T, Carrasco R, et al. Identification of genes modulated in multiple myeloma using genetically identical twin samples. Blood 2004;103:1799-1806.

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Predicting Outcomes in CLL

Michael Williams, MD

Dr. Williams indicated no relevant conflicts of interest.

Grever MR, Lucas DM, Dewald GW, et al. Comprehensive assessment of genetic and molecular features predicting outcome in patients with chronic lymphocytic leukemia: results from the US Intergroup phase III trial E2997. J Clin Oncol 2007;25:799-804.

CLL is a highly heterogeneous disorder with markedly disparate clinical course and variable therapeutic response among patients. A number of phenotypic and molecular markers have been shown in retrospective studies to correlate with the pace of disease progression, time to first treatment, and treatment response. In this paper, Grever and colleagues prospectively analyzed CLL cells from patients in the U.S. Intergroup trial (ECOG/ CALGB/SWOG) of fludarabine (F) single agent vs. fludarabine plus cyclophosphamide (FC) as initial therapy for symptomatic patients who require treatment. Pre-treatment samples were analyzed for IgVH (immunoglobulin heavy chain variable gene) and p53 mutation status, expression of ZAP-70, CD38 and other phenotypic markers, and cytogenetics by FISH (fluorescent in situ hybridization). The study demonstrated significantly improved complete and overall response rates in patients receiving FC, as detailed in an accompanying report¹. Two hundred and thirty-five of the 278 study patients had the correlative marker analyses performed. None of the markers correlated with response to either treatment arm. However, patients with the del(17p13.1) or del(11q22.3) had a significantly shorter progression-free survival (PFS) with either F or FC therapy. Interestingly, mutations in the p53 gene located at the chromosome 17p locus did not correlate with outcome, suggesting another relevant gene or genes in the deleted segment.

The clinical and biologic heterogeneity of CLL, reflected in definable subsets of disease using molecular and phenotypic markers, has led to the hypothesis that a risk-adapted therapeutic approach may be feasible. This study by Grever et al. is among the first to prospectively apply these markers in a randomized multicenter trial. The results confirm a significantly shorter PFS for patients with del(17p) or del(11q) despite similar responses to induction therapy. Such high-risk patients would be logical candidates for alternative initial treatment or consolidation approaches. It will be important to validate these findings in ongoing and future CLL clinical trials, and to standardize methodologies and endpoints for positive vs. negative marker expression. The predictive value for any marker may well differ depending upon the therapeutic regimen applied, as shown by a recent study wherein CLL patients with the del(11q) responded as well to the PCR regimen (pentostatin, cyclophosphamide, rituximab) as did those with more favorable markers². The use of prognostic markers thus will be a moving target, but nonetheless one that helps us know where to aim.

1. Flinn IW, Neuberg DS, Grever MR, et al. Phase III trial of fludarabine plus cyclophosphamide compared with fludarabine for patients with previously untreated chronic lymphocytic leukemia: US Intergroup Trial E2997. J Clin Oncol 2007;25:793-8.
2. Kay NE, Geyer SM, Call TG, et al. Combination chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab shows significant clinical activity with low accompanying toxicity in previously untreated B chronic lymphocytic leukemia. Blood 2007;109:405-11.

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Rituximab in ITP – When and Why Does It Work?

Charles Abrams, MD

Dr. Abrams indicated no relevant conflicts of interest.

Arnold DM, Dentali F, Crowther MA, et al. Systematic review: efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura. Ann Intern Med 2007;146:25-33.

Despite the increasing use of rituximab in the treatment of idiopathic thrombocytopenic purpura (ITP), there is a remarkably small amount of literature describing its efficacy and toxicity. The authors of this study reviewed all of the articles on this topic published between 1997 and 2004 and found only 19 reports that described five or more patients (313 total patients). Of these 19 reports, nine were published only in abstract form. The median duration of followup was only 9.5 months (range: two to 25 months). All of the publications were case reports, case series, or single-arm cohorts, and there were no randomized trials. The reported response rates were higher in studies that contained small numbers of patients, making definitive conclusions about the response rate difficult.

Pre-treatment platelet counts varied between 1,000 – 89,000/µl. Almost all of the patients had been previously treated with corticosteroids, and approximately half of the patients had failed a splenectomy. Patients were treated with four weekly doses of 375mg/m2 in 16 of the 19 studies, while the remainder did not state the dose, or varied either the dose or the duration of therapy. The overall response rate (platelet count >50,000) was 63 percent, and the complete response rate (platelet count >150,000) was 46 percent. The median duration of a response was 10.5 months (range: three to 20 months). Although the median time to a response was 5.5 weeks, it was curiously as short as two weeks in some series (more on this topic later). The majority of the treatment-induced toxicity was infusion-related serum sickness, or other allergic responses. Nine deaths were reported, and most of these events were hemorrhagic or due to an underlying comorbidity. Several patients died of infections, but not clearly related to treatment.

Splenectomy has long been considered the gold standard of therapy for patients who require treatment for chronic ITP. It produces long-term response rates of 65-70 percent, and many of the remaining patients derive some benefit from the procedure. Although splenectomy has an impressive response rate, it is associated with approximately 1 percent operative mortality, as well as a lifelong increased risk of opportunistic infections. The alternative standard therapy is chronic immunosuppressive drug therapy.

The monoclonal antibody against CD20-positive B cells, rituximab, has received recent enthusiasm as an immunosuppressive agent for a wide variety of autoimmune diseases. It is clear that rituximab can induce remissions in ITP. Although, it has been difficult to estimate its true efficacy since the literature is biased by small case series touting high response rates. By restricting analysis to publications that report response rates based on more than five patients, Arnold and colleagues identified only 10 papers and nine abstracts that focused on rituximab efficacy in ITP. Their results place the response rate of rituximab in the same category, but probably not better than most other immunosuppressive agents. However, rituximab still does have the advantage of a relatively good safety profile.

Does this mean that rituximab is a reasonable alternative treatment for ITP patients who require chronic therapy? Although it is being used with increasing frequency as hematologists become more familiar with the drug, we really cannot state definitive efficacy rates or long-term toxicity data to our patients as we can for splenectomy treatment. It is also unclear how anti-B-cell therapy for ITP can sometimes produce a response within a few days. Circulating IgG has a half-life of approximately three weeks, so if rituximab's effect is to stop all additional immunoglobulin production, its response should not be apparent for several weeks to months. One possible explanation for the rapid effect is that rituximab-coated B cells may compete with immunoglobulin-coated platelets by the reticuloendothelial system¹. However, it is surprising that enough antibody-coated B cells are generated after rituximab therapy to mimick a "WinRho-like" effect. If this model for the rapid rituximab effect is correct, then one would predict that it would not be seen in post-splenenctomy patients. Thus far, this does not appear to be the case.

Although rituximab is clearly a useful agent in the treatment of ITP, the published data is currently a little thin. It would therefore be worthwhile and beneficial for both patients and physicians to study rituximab in a larger cohort. Only then will we be able to definitively understand the efficacy and toxicity of this therapy for ITP.

1. Bennett CM, Rogers ZR, Kinnamon DD, et al. Prospective phase 1/2 study of rituximab in childhood and adolescent chronic immune thrombocytopenic purpura. Blood 2006;107:2639-42.

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Expansion Hopes Within Reach?

Nelson Chao, MD

Dr. Chao indicated no relevant conflicts of interest.

Serafini M, Dylla SJ, Oki M, et al. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells. J Exp Med 2007;204:129-39.

The bone marrow is a remarkable organ. Every time your heart beats, millions of cells are created. The turnover of these cells requires a tightly regulated balance between the formation of all these blood cells and their senescence and death. Our bodies are able to choreograph this birth and death routine in part due to stem cells, which differentiate and self-renew with each division. The characterization of hematopoietic stem cells has been a remarkably fruitful area of research. Our bone marrow contains at least two types of stem cells, the hematopoietic stem cell (HSC) and a stromal stem cell that is able to give rise to cartilage, adipose tissue, muscle, and bone. One area of exciting research has been the identification of multipotent adult progenitor cells (MAPCs), which could be coaxed from the bone marrow and which are thought to be the precursors for the non-hematopoietic cells. In this paper, Catherine Verfaillie’s laboratory describes convincing data demonstrating that these MAPCs can be expanded in vitro for more than 40-80 population doublings without senescence and that these cells are capable of multilineage engraftment in immunodeficient mice, although approximately 1000-fold more MAPCs were required compared to purified HSC. The HSC derived from these MAPCs are able to be serially transplanted into secondary and tertiary recipients and the lymphocytes derived from these MAPCs were fully functional. Moreover, using green fluorescent protein transgenic mice and congenic mice, the data suggests that fusion did not occur.

One of the many holy grails in hematology is the ability to expand hematopoietic stem cells. This goal has been elusive in that when bone marrow is placed in culture, there can be a dramatic increase in committed precursors, but there seems to be a limit in the number of true HSCs that expand. Consequently, when these expanded cells are returned to the recipient, there is little beneficial clinical effect that can be observed. These MAPCs, in contrast, seem to be able to expand for prolonged periods ex vivo without evidence of senescence. The promise of these MAPCs is that they can contribute robustly to hematopoiesis, generating myeloid and lymphoid cells that can be serially transplanted. Moreover, no tumor formation was observed, which will be important in therapeutic applications. The immediate applications include expansion of HSCs for bone marrow reconstitution or the use of these cells to induce transplantation tolerance. It is not clear yet how these adult MAPCs will function compared to embryonic stem cells or whether these cells will be able to be more broadly used for regenerative medicine outside of hematopoiesis. However, for the time being, the possibility of a readily expandable population of cells that can make blood is truly exciting.

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Enhancing Hematopoietic Function with Parathyroid Hormone

Diane Krause, MD, PhD

Dr. Krause indicated no relevant conflicts of interest.

Adams GB, Martin RP, Alley IR, et al. Therapeutic targeting of a stem cell niche. Nat Biotechnol 2007;25:238-243.

This study investigates the potential clinical utility of expanding the hematopoietic stem cell (HSC) niche in the bone marrow (BM) with parathyroid hormone (PTH). Although still controversial, several lines of evidence suggest that osteoblasts support HSC expansion and/or survival in the BM, and therefore comprise at least part of the HSC niche. HSCs are found in close association with osteoblasts, osteoblasts support progenitor cells in in vitro BM cultures¹, selective depletion of osteoblasts from adult mice causes a decrease in HSC in the BM², and interventions that increase bone trabecular surface area in mouse BM increase HSC numbers^3,4. The laboratory of Dr. David Scadden published previously that in vivo administration of PTH leads to increased osteoblast numbers, increased bone trabeculae, and an increase in the number of HSC³. Furthermore, PTH administration enhanced survival in a mouse BMT model in which suboptimal numbers of HSC were transplanted. These exciting findings suggest that PTH could provide therapeutic benefit to patients undergoing BMT.

The discovery that PTH stimulates bone formation is not new, but this anabolic function is a bit counterintuitive. The role of PTH in vivo is to increase plasma calcium levels by promoting calcium release from the bone, increasing calcium absorption in the GI tract, and increasing renal tubular calcium resorption. Consistent with its effect to induce release of calcium from bone, continuous administration of full-length (84 amino acid) PTH leads to bone loss and can cause osteoporosis. In contrast, intermittent PTH administration leads to bone formation. Furthermore, the 34 aa amino terminus of PTH maintains the anabolic activity of full-length PTH without causing bone loss. The PTH amino-terminal fragment, known as teriparatide, has been used in promising clinical trials for treating osteoporosis, and was used in this study.

Three clinically relevant scenarios were tested in this manuscript using murine models. First, PTH was administered to test for enhancement of mobilization. Pre-treatment with PTH led to a two-fold increase in the number of HSC in the peripheral blood with no effect on the WBC. These findings suggest that PTH can enhance PBSC mobilization by expanding the BM HSC pool. The investigators next tested whether PTH could prevent the depletion/exhaustion of HSC that can occur with repeated rounds of chemotherapy followed by G-CSF to promote WBC recovery. Mice were exposed to five rounds of cytoxan, each of which was followed by treatment with G-CSF alone or G-CSF plus PTH. After this regimen, PTH led to an increase in the number of HSC in the BM and the number of HSC that could be mobilized into the peripheral blood in response to G-CSF, suggesting that PTH can protect the marrow from the HSC depletion that can occur with repeated rounds of chemotherapy. The third clinical question addressed was really a continuation of this group’s previous studies. The investigators had already shown that PTH administration enhanced survival after lethal radiation and administration of low doses of HSC. Here they proved that this effect of PTH was mediated, at least in part, via its ability to promote (indirectly) HSC self-renewal.

Clinical studies are underway to determine whether PTH can promote engraftment in adult recipients of cord blood transplants. Clinical trials using PTH in the setting of BMT for lymphoma are also being initiated. Perhaps future studies will reveal other therapeutic indications for PTH. There are other features of the HSC niche that could be targeted. For example, the sinusoidal endothelium in the BM also supports HSC survival/proliferation, and enhancement of these cells may also provide important clinical benefits. Pharmacological agents could also be targeted to pathways that regulate HSC interactions with the niche, such as those stimulated by notch, N-cadherin, and/or angiopoietin.

1. Taichman RS, Emerson SG. Human osteoblasts support hematopoiesis through the production of granulocyte colony-stimulating factor. J Exp Med 1994;179:1677-1682.
2. Visnjic D, Kalajzic Z, Rowe DW, et al. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 2004;103:3258-3264.
3. Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425:841-846.
4. Zhang J, Niu C, Ye L, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425:836-841.

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