Femoral Marrow Research Articles

Abstract 2229: CD133+/CD44+ Cancer Stem Cells Represent a Disproportionately Large Fraction of Early Bone Disseminated Prostate Cancer Tumor Cells

Abstract Introduction: Nearly 30% of prostate cancer patients presenting with organ confined disease experience biochemical recurrence within 10 years of curative prostatectomy. A recent analysis of bone marrow aspirates reported the presence of disseminated tumor cells (DTCs) in 57% of patients with no evidence of disease following prostatectomy, suggesting that early bone homing prostate cancer DTCs may contribute to disease recurrence. Little is currently known about the biology of these cells. Procedures: Murine models were developed to isolate and study early bone homing prostate cancer disseminated tumor cells. Early dissemination was modeled through intracardiac injection of 1 × 106 prostate cancer cells of the PC3 cell line into SCID mice 15-24 hours prior to euthanasia. Spontaneous early tumor cell dissemination was modeled using subcutaneous prostate cancer tumors formed in SCID mice by injecting 5 × 105 DU145 cells in matrigel three weeks prior to euthanasia. Bone marrow was flushed from the femurs of euthanized mice and depleted of cells of hematopoietic-lineage using the Lineage Cell Depletion Kit (Miltenyi Biotec, 130-092-211). DTCs were identified in lineage depleted murine marrow using a FITC conjugated antibody for human leukocyte antigen (>90% of PC3 and DU145 cells are HLA-ABC+) and isolated by fluorescence-activated cell sorting (FACS). Cell surface expression of the cancer stem cell markers CD133 and CD44 on bone homing DTCs were analyzed by flow cytometry. Results: Prostate cancer DTCs were detected in murine bone marrow, representing 3.1% ± 0.9% (n=4-mice) and 1.2% ± 0.2% (n=4-mice) of lineage depleted femoral marrow cells from the intracardiac and subcutaneous models, respectively. Early bone disseminated PC3 cells in the intracardiac injection model demonstrated a large fraction of CD133+/CD44+ cancer stem cells (25.3% ± 4.2%, n=4). This is substantially greater than the 0.73% fraction found with the injected PC3 cells. Spontaneous early disseminated bone homing DTCs from subcutaneous DU145 tumors demonstrated similar results. A large fraction of DTCs were positive for both stem cell markers (17.3% ± 3.6%, n=4), considering that the injected DU145 cells only had a 0.28% fraction of CD133+/CD44+ cells. Conclusions: Early bone disseminated prostate cancer cells in murine xenograft models can be enriched by lineage depletion, identified by human leukocyte antigen, and subsequently isolated and characterized by flow cytometry. Early prostate cancer bone disseminations in both intracardiac injection and subcutaneous tumor models have a disproportionately large fraction of cancer stem cells identified using the surface markers CD133 and CD44. This suggests a proclivity for bone dissemination by CD133+/CD44+ prostate cancer stem cells and a possible role for these cells as DTCs responsible for biochemical failure after curative prostatectomy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2229.

CD36 Is a Novel Serum Amyloid A (SAA) Receptor Mediating SAA Binding and SAA-induced Signaling in Human and Rodent Cells

Serum amyloid A (SAA) is a major acute phase protein involved in multiple physiological and pathological processes. This study provides experimental evidence that CD36, a phagocyte class B scavenger receptor, functions as a novel SAA receptor mediating SAA proinflammatory activity. The uptake of Alexa Fluor 488 SAA as well as of other well established CD36 ligands was increased 5-10-fold in HeLa cells stably transfected with CD36 when compared with mock-transfected cells. Unlike other apolipoproteins that bind to CD36, only SAA induced a 10-50-fold increase of interleukin-8 secretion in CD36-overexpressing HEK293 cells when compared with control cells. SAA-mediated effects were thermolabile, inhibitable by anti-SAA antibody, and also neutralized by association with high density lipoprotein but not by association with bovine serum albumin. SAA-induced cell activation was inhibited by a CD36 peptide based on the CD36 hexarelin-binding site but not by a peptide based on the thrombospondin-1-binding site. A pronounced reduction (up to 60-75%) of SAA-induced pro-inflammatory cytokine secretion was observed in cd36(-/-) rat macrophages and Kupffer cells when compared with wild type rat cells. The results of the MAPK phosphorylation assay as well as of the studies with NF-kappaB and MAPK inhibitors revealed that two MAPKs, JNK and to a lesser extent ERK1/2, primarily contribute to elevated cytokine production in CD36-overexpressing HEK293 cells. In macrophages, four signaling pathways involving NF-kappaB and three MAPKs all appeared to contribute to SAA-induced cytokine release. These observations indicate that CD36 is a receptor mediating SAA binding and SAA-induced pro-inflammatory cytokine secretion predominantly through JNK- and ERK1/2-mediated signaling.

Hydrolyzed collagen improves bone metabolism and biomechanical parameters in ovariectomized mice: An in vitro and in vivo study

Collagen has an important structural function in several organs of the body, especially in bone and cartilage. The aim of this study was to investigate the effect of hydrolyzed collagen on bone metabolism, especially in the perspective of osteoporosis treatment and understanding of its mechanism of action. An in vivo study was carried out in 12-week-old female C3H/HeN mice. These were either ovariectomized (OVX) or sham-operated (SHAM) and fed for 12 weeks with a diet containing 10 or 25 g/kg of hydrolyzed collagen. We measured bone mineral density (BMD) using dual-energy X-ray absorptiometry (DXA). C-terminal telopeptide of type I collagen (CTX), marker of bone resorption, and alkaline phosphatase (ALP), marker of bone formation, were assayed after 4 and 12 weeks. Femur biomechanical properties were studied by a 3-point bending test and bone architecture by microtomography. The BMD for OVX mice fed the diet including 25 g/kg of hydrolyzed collagen was significantly higher as compared to OVX mice. The blood CTX level significantly decreased when mice were fed with either of the diets containing hydrolyzed collagen. Finally, we have shown a significant increase in bone strength correlated to geometrical changes for the OVX mice fed the 25 g/kg hydrolyzed collagen diet. Primary cultures of murine bone cells were established from the tibia and femur marrow of BALB/c mice. The growth and differentiation of osteoclasts and osteoblasts cultured with different concentrations (from 0.2 to 1.0 mg/mL) of bovine, porcine or fish hydrolyzed collagens (2 or 5 kDa) were measured. Hydrolyzed collagens (2 or 5 kDa) in the tissue culture medium did not have any significant effects on cell growth as compared to controls. However, there was a significant and dose-dependent increase in ALP activity, a well-known marker of osteogenesis, and a decrease in octeoclast activity in primary culture of bone cells cultured with hydrolyzed collagens (2 kDa only) as compared to the control.It is concluded that dietary hydrolyzed collagen increases osteoblast activity (as measured in primary tissue culture), which acts on bone remodeling and increases the external diameter of cortical areas of the femurs.

CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK

The mechanisms regulating the migration of leukaemic cells between the blood and bone marrow compartments remain obscure, but are of fundamental importance for the dissemination of the disease. This study investigated the in vivo homing of human B cell progenitor acute lymphoblastic leukaemia (ALL) cells to the femoral bone marrow of non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. It was demonstrated that patient ALL cells use the chemokine axis, chemokine (CXC motif) receptor 4 (CXCR4)/ chemokine (CXC motif) ligand 12 (CXCL12), to home to the femoral marrow. CXCL12-mediated signalling through p38 mitogen-activated protein kinase (MAPK) was required for optimal homing. In contrast, the homing of normal peripheral blood CD34(+) cells and the cytokine-dependent CD34(+) cell line Mo7e was independent of p38MAPK, consistent with the dependence of these cells, as well as normal CD34(+) CD19(+) B cell progenitors, on PI-3K/AKT signalling. Altogether, our data provide clarification of the direct role of CXCL12 in the bone marrow homing of ALL cells and demonstrate unique signalling molecule usage that may have therapeutic implications for this disease.

Increased Bone Marrow Fat in Anorexia Nervosa

Although women with anorexia nervosa (AN) have severe depletion of body fat, a paradoxical increase in bone marrow fat has been described. Recent data suggest that marrow fat measured by 1H-magnetic resonance spectroscopy (MRS) in combination with bone mineral density (BMD) may be more valuable than either parameter alone in detecting bone weakness. The objective of the study was to investigate the effect of AN on accumulation of marrow fat in spine and femur using 1H-MRS and the relationship between marrow fat, BMD, and body composition in subjects with AN and normal-weight controls. This was a cross-sectional study. The study was conducted at a referral center. Patients included 10 women with AN (29.8 +/- 7.6 yr) and 10 normal-weight age-matched women (29.2 +/- 5.2 yr). There were no interventions. Marrow fat content of the fourth lumbar vertebra and femur measured by 1H-MRS. BMD of spine and hip measured by dual-energy x-ray absorptiometry. Subjects with AN had higher marrow fat at the fourth lumbar vertebra and femur compared with controls (P = 0.004-0.01). There was an inverse correlation between marrow fat of L4 and femur and BMD of the spine and hip (r = -0.56 to -0.71, P = 0.01-0.0002) and body mass index and sc adipose tissue of the thigh (r = -0.49 to -0.71, P = 0.03-0.0007). There was an inverse correlation between femur marrow fat and sc and total abdominal adipose tissue (r = -0.53 to -0.67, P = 0.003-0.03). Women with AN have greater lumbar and femoral marrow fat than controls, and marrow fat correlates inversely with BMD. This paradoxical increase in marrow fat at a time when sc and visceral fat are markedly reduced raises important questions about functional consequences of this process.

Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

Gene Transfer Using Recombinant Simian Virus 40 Viral Vectors Into Mice Bone Marrow Progenitor Cells Depressed the Immunogenicity of Keratinocyte Stem Cells

Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Herein we have attempted to overcome these limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they efficiently transduce resting CD34+ cells. Neonatal C57BL/6 (H-2b) mice (3 days old) received SV(Nef-FLAG), carrying FLAG marker epitope directly into both femoral marrow cavities. Keratinocyte stem cells (KSCs) were purified at 7 and 14 days after SV40 injection. The KSCs from 10-day-old C57BL/6 mice were designated as controls. Flow cytometric (FCM) analyses indicated that KSCs from transgenic mice showed strong down-regulation of surface immunological molecules CD40, CD80, CD86, and human major histocompatibility complex class I chain-related antigen A (MICA). Mixed lymphocyte reaction (MLR) assays showed that transgenic KSCs depressed allogeneic T-cell proliferation. Immunofluorescence showed transgenic KSCs expressed FLAG for the entire study as well as high levels of transforming growth factor (TGF)-β and BCL-2. Thus, direct intramarrow administration of recombinant SV40 yielded efficient gene transfer to mice BM progenitor cells. KSCs with low immunogenicity may be obtained for further investigations of skin transplantation immunity.

Pomalidomide Augments Erythropoiesis and Fetal Hemoglobin Production in a Humanized Mouse Model of Sickle Cell Disease

Introduction: Pomalidomide (PL) is an IMiD™ immunomodulatory compound. Thalidomide and lenalidomide have previously been shown to mitigate blood transfusion dependency in patients with thalassemia major and myelodysplastic syndromes, respectively.1,2 In a recent in vitro study, PL, which is currently being evaluated for the treatment of hematological cancers, stimulated erythropoiesis, F-cell production, total hemoglobin (Hb) and fetal hemoglobin (HbF) synthesis in human CD34+ cells.3 Furthermore, combined treatment of CD34+ cells with PL and Hydroxyurea (HU) synergistically upregulated HbF expression. These results suggest a promising role for PL in the treatment of β-hemoglobinopathies. To evaluate the in vivo efficacy of PL in sickle cell disease (SCD), we conducted an eight week study in a relevant knockout-transgenic (KT) mouse model. 4Methods:Animals. Six week old KT homozygous sickle mice were treated daily (Mon–Fri; i.p. injections) for eight weeks with the following compounds: Grp 1. Vehicle (n=8); Grp 2. PL (10 mg/kg; n=9); Grp 3. HU (100 mg/kg; n=7); Grp 4. PL+ HU (n=8). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes. Complete blood count (CBC) was analyzed with the CBC-Diff™ Veterinary Hematology System (Heska Inc., Loveland, CO). Reticulocyte counts were determined by supravital staining with methylene blue. HbF analysis of mouse hemolysates was done by HPLC using a weak cation-exchange column SynChropak CM-300 (Elchrom INC, Darien, IL). Organ analysis (liver, spleen, and femur marrow) included organ weights (%bw) and histology of H&E paraffin sections.Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant.Results:Activity level and social behavior were unaffected by the treatment groups. There were no physical signs of drug toxicity and weight gains were comparable for all groups.PL significantly augmented HbF expression comparable to HU (HbF [peak%]: Veh: 6.24±0.35; PL: 9.51±0.83, P<0.01; HU: 10.54±0.77, P<0.01/total Hb [gm/dl]: Veh: 7.16±0.38; PL: 7.67±0.33; HU: 6.09±0.79). Surprisingly, PL-induced HbF production returned to control values after combinatory therapy with HU (HbF [peak%]: Veh: 6.24±0.35; PL+HU: 7.14±0.32). In contrast to HU, PL enhanced erythropoiesis as evidenced by increases in total RBCs, reticulocytes, and spleen weight (RBCs [×106/μl]: Veh: 4.84±0.14; PL: 5.49±0.19, P<0.05; HU: 4.47±0.51/Retics [%]: Veh: 36.81±5.78; PL: 40.47±4.73; HU: 16.41±3.94, P=0.01/Spleen weight [%bw]: Veh: 4.07±0.27; PL: 4.92±0.2, P=0.02; HU: 2.75±0.25, P<0.01). The total WBC count was largely unaffected by PL, but decreased significantly with HU (WBC [×103/μl]: Veh: 14.43±2.74; PL: 11.62±2.62; HU: 6.85±0.82, P<0.05).The PL group showed a trend toward increased marrow hyperplasia. A detailed analysis of cell density, erythroid and myeloid cells is underway. Liver histology revealed decreased tissue inflammation and focal necrosis in ~50% of PL-treated animals.Summary & Conclusions: 1. PL caused a robust induction of HbF in sickle mice. These results extend earlier in vitro findings of PL's bioactivity in human erythroid progenitor cells. The HbF response to PL was similar to the established HbF-inducer HU, but surprisingly was lost in combination with HU. This inhibitory effect suggests that PL and HU stimulate HbF by distinct mechanisms. 2. Additionally, PL augmented erythropoiesis as demonstrated by increases in total RBCs, reticulocytes, spleen weight, and marrow hyperplasia. In contrast, HU showed the expected findings of generalized myelosuppression. 3. PL reduced liver injury and inflammation in ~50% of treated animals. Liver preservation tended to be associated with higher HbF values, though PL's immunomodulatory properties may have contributed to this outcome. 4. In summary, PL exhibits a highly favorable hematological profile in a humanized mouse model of SCD. These results warrant further investigation in a Phase I trial of patients with SCD.

Co-Transplantation of MSC Improves the Engraftment of HSC after Auto-iBMT in Non-Human Primates.

[Background] Mesenchymal stem cells (MSC) have been shown to play critical roles in various in vivo phenomena including osteoblastic differentiation. It has been suggested that, in the bone marrow, hematopoietic stem cells (HSC) reside in osteoblastic niche, which consists of osteoblasts derived from MSC. In mice, previous studies have demonstrated that co-transplantation of MSC improves the engraftment of HSC, especially after transplantation of the cells into the bone marrow cavity directly, namely intra-bone marrow transplantation (iBMT). However, neither the efficacy nor the dynamics such as migration and homing of HSC after iBMT with MSC have been determined in large animals. Here, using non-human primates, we have investigated the effects of co-transplantation of MSC on the engraftment of HSC after autologus iBMT.[Methods] Auto-iBMT of cynomolgus monkeys was performed, using bone marrow stromal cells (as MSC) and CD34-positive cells (as HSC). The latter were divided into two equal aliquots, each of which was genetically marked with a different retroviral vector, G1Na or LNL6. Conditioning of iBMT, TBI or administration of busulfan, was followed by hemi-iBMT; that is, the bone marrow of one side (right or left) of the body was transplanted with one HSC aliquot together with MSC, whereas the other side of the identical body was transplanted with the other HSC aliquot alone. Engraftment of each HSC aliquot was evaluated by colony PCR of bone marrow, as well as by PCR of the genomic DNA obtained from peripheral blood or bone marrow of humerus, femur, and ilium. Both PCR could distinguish the dual markings derived from the two HSC aliquots.[Results] In the first monkey transplanted, we found that the engraftment derived from the co-transplantation aliquot was 4.4-times higher than that derived from the HSC alone aliquot as assessed by colony PCR (48% versus 11%) using the bone marrow samples obtained from the ilium at day 46 post-iBMT. In the second monkey, when the peripheral WBC recovered to 2500–3000/μl after day 28 post-iBMT, 2% of the cells were positive with the retroviral marking derived from the co-transplantation aliquot, although none of them were positive with that derived from the HSC alone aliquot. In addition, colony PCR of the humerus and femur of both sides at day 39 post-iBMT revealed that the engraftment derived from the co-transplantation aliquot was 6.0-times higher than that derived from the HSC alone aliquot. Notably, colony-forming units (CFU) derived from the cotransplantation aliquot were detected in the bone marrow of the opposite side, suggesting that HSC injected into the bone marrow might migrate and achieve homing in the distant bone marrow.[Conclusion] Taken together, these results indicate that, in auto-iBMT of cynomolgus monkeys, co-transplantation of MSC improves the engraftment of HSC, the efficacy of which might be attributable to additional osteoblastic niche, presumably created from co-transplanted MSC.

The Kinase Activity of IL-1 Receptor-associated Kinase 4 Is Required for Interleukin-1 Receptor/Toll-like Receptor-induced TAK1-dependent NFκB Activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.

Bioactivity of a Novel Nano— composite of Hydroxyapatite and Chitosan—Phosphorylated Chitosan Polyelectrolyte Complex

The bioactivity of a novel composite of carbonate-containing low-crystallinity nanoparticle hydroxyapatite (HA) and a chitosan—phosphorylated chitosan polyelectrolyte complex (PEC) was evaluated in vitro and in vivo. The HA—PEC nanocomposite with complicated porous structure was prepared by a biomimetic method. An acidic chitosan (polycation) solution containing calcium and phosphate ions (6 mM Ca2+, Ca/P: 1.67) was added into phosphorylated chitosan (polyanion) solution; the formation of PEC and the controlled HA crystal growth were co-organized in alkaline solution. The material was co-cultured with rat osteoblasts in vitro, and implanted into rabbit femur marrow cavities. The results indicate that the PEC—HA composite promoted osteoblast adhesion, morphology, proliferation, and differentiation in vitro; the bone tissue response to the material histologically showed that it was bioactive, as well as biodegradable. The HA—PEC composite shows promise as a bone-repair material.

Isolation, characterisation and osteogenic potential of human bone marrow stromal cells derived from the medullary cavity of the femur

Marrow stromal cells (MSC) are increasingly being introduced in orthopaedic practice as potentially powerful effectors of bone regeneration. Since cell recovery of MSC is affected by a high degree of individual variability, sources for collecting adequate amounts of safe and effective MSC under routine conditions are needed. We analysed if femoral bone marrow, which is usually discarded during total hip arthroplasty procedures, is a reliable source of MSC to enhance bone healing and regeneration. Mononuclear cells were isolated, assayed for typical MSC markers, harvested under appropriate culture conditions and evaluated for their ability to differentiate into osteoblasts. Cell recovery and osteogenic potential were independent from donor gender or age, suggesting that elderly individuals are eligible for autologous cell therapy. Although heterogeneous, the pool of MSC recovered from femoral marrow without further in vitro selection or manipulation proved highly effective in proliferating and differentiating along the osteogenic lineage. In conclusion, this source of MSC offers a valuable tool to be used to promote osteogenesis and implant fixation.

Bone marrow MR imaging as predictors of outcome in hemopoietic stem cell transplantation

The purpose of this study is to investigate the role of femoral marrow MR imaging as predictor of outcome for hemopoietic stem cell transplantation (HSCT) in beta-thalassemia major. MR imaging of the proximal femur, including T1- and T2-weighted spin echo and short-tau inversion recovery and in-phase and out-of-phase fast field echo images, was prospectively performed in 27 thalassemia major patients being prepared for HSCT. The area of red marrow and its percentage of the proximal femur were measured, and the presence of marrow hemosiderosis was assessed. Age-adjusted multivariate logistic regression was used to determine the relationship between red marrow area percentage and marrow hemosiderosis and HSCT outcome. Red area percentage were less in patients with successful (90.25 +/- 4.14%) compared to unsuccessful transplants (94.54% +/- 2.93%; p = 0.01). Red marrow area percentage correlated positively with duration of symptoms(r = 0.428, p = 0.026) and serum ferritin (r = 0.511, p = 0.006). In multivariate-adjusted logistic regression analyses, red marrow area percentage was significantly inversely associated with successful HSCT (OR = 1.383, 95% CI: 1.059-1.805, p = 0.005). Marrow hemosidersosis and duration of sympotms and serum ferritin were not associated with HSCT outcome(p = 0.174, 0.974, 0.762, respectively). Red marrow area percentage of proximal femur on MR imaging is a useful predictor of HSCT outcome.

Serum FLT‐3 ligand in a busulphan‐induced model of chronic bone marrow hypoplasia in the female CD‐1 mouse

The concentration of the cytokine fms-like tyrosine kinase-3 ligand (FL) is elevated in the plasma of patients treated with chemotherapy or radiotherapy for malignant conditions. In addition, plasma FL is increased in patients with bone marrow failure resulting from stem-cell defects (e.g. aplastic anaemia). Our goal in the present study was to measure the concentration of serum FL in mice treated with the chemotherapeutic agent busulphan (BU) to induce bone marrow depression and relate changes in FL to effects on haemopoiesis. Female CD-1 mice were treated with BU (9.0 mg/kg) or vehicle by intraperitoneal injection on 10 occasions over 21 days. Animals were autopsied on days 1, 23, 72, 119 and 177 postdosing. A full blood count was performed, and serum prepared for FL analysis. Femoral marrow cell suspensions were prepared to assess the total femoral nucleated cell count (FNCC) and the number of committed haemopoietic progenitor cells (CFU-C). On days 1 and 23 postdosing, significant decreases were evident in many peripheral blood parameters; the FNCC and CFU-C were also reduced in BU-treated mice, in conjunction with increases in serum FL levels. On days 72, 119 and 177 postdosing, several peripheral blood and bone marrow parameters remained reduced and the concentration of serum FL continued to be significantly increased. Linear regression analysis demonstrated significant correlations between the concentration of serum FL in BU-treated mice and peripheral blood and bone marrow parameters; this suggests the possible use of serum FL as a potential biomarker for drug-induced bone marrow injury.

The haemotoxicity of azathioprine in repeat dose studies in the female CD-1 mouse.

Azathioprine (AZA) is a cytotoxic immunosuppressive drug used in the prevention of rejection in organ transplants and the treatment of auto-immune diseases. However, AZA is haemotoxic causing significant bone marrow depression. The present studies were to characterize the haemotoxicity of AZA in the female CD-1 mouse. In Experiment 1, a dose-ranging study, with AZA gavaged daily for 10 days, clinical evidence of toxicity was evident at 125 mg/kg and above. Experiment 2 was a dose-response study with AZA gavaged daily for 10 days at 40-120 mg/kg. At day 1 after the final dose, AZA induced a dose-related pancytopaenia, reduced femoral marrow cellularity, increases in serum levels of the cytokine fms-like tyrosine kinase 3 ligand, reduction in granulocyte-monocyte colony-forming units and erythroid colonies, and increased bone marrow apoptosis. Histology demonstrated hepatocyte hypertrophy, thymic atrophy, reduced splenic extramedullary haemopoiesis, and reduced cellularity of sternal bone marrow. In Experiment 3, AZA was dosed for 10 days at 100 mg/kg with autopsies at 1, 3, 9, 22, 29, 43 and 57 days postdosing. At 1, 3 and 9 days, haematological parameters reflected changes in Experiment 2. At 22/29 days, many blood parameters were returning towards normal; at 43/57 days, most parameters compared with controls. However, there was some evidence of a persistent (i.e. residual/late-stage) mild reduction in RBC and erythroid progenitor cell counts at day 43/57. We conclude that the CD-1 mouse provides an acceptable model for the haemotoxicity of AZA in man.

Initial study of optimal single-voxel 1H-MR spectroscopy parameters on femoral bone marrow

目的 选择符合股骨骨髓腔解剖学特点、检查时间短、重复性良好而且图像质量高的MR氢质子波谱(1H-MRS)采集方法.方法 采用1.5 T MR单体素波谱激发回波探测序列对26名志愿者正常股骨骨髓进行1H-MRS的探索性研究.其中12名股骨1H-MRS采用128次采集、1 cm×1 cm×1 cm大小的立方形感兴趣容积(VOI)在同一受检部位重复采集2～3次.另14例股骨1H-MRS分别采用128次和256次采集、1 cm×1 cm×1 cm和2 cm×2 cm×2 cm大小VOI.结果 采用1 cm×1 cm×1 cm大小的VOI和128次采集获得的1H-MRS基线稳定,水半高宽≤8～12 Hz,信噪比为11.31,2～3次重复采集后谱线可重复率为100%.不同部位采集的1H-MRS表现不尽相同,最大波峰位置位于0.90 ppm(×10-6)或1.65 ppm附近处.但同一部位采用不同的VOI大小和不同的采集次数所获得的谱线全部相同或相似.1H-MRS采集时间与VOI大小无关,128次和256次采集时间分别为199 s和391 s.结论 采用1 cm×1 cm×1 cm大小的VOI和128次采集股骨骨髓1H-MRS具有临床可行性,采集时间较短,图像质量高而且重复性良好,具有较强的临床实用性。

InVitro Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Interfered with Their Distribution to Other Bone Marrow Compartments after Intra-Bone Marrow Transplantation.

Intra-bone marrow transplantation (IBMT) is a novel strategy for transplantation of hematopoietic stem cells because it can transfer various types of cells to bone marrow regardless of their homing capacity. However, reconstitution process of these cells after IBMT remains to be fully elucidated. Here, we investigated whether in vitro culture of cord blood hematopoietic stem/progenitor cells affects their reconstitution in bone marrow after IBMT. Freshly isolated AC133+ cells (5x104 cells/mouse) or all cells derived from AC133+ cells cultured with growth factors (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation was not considerably different between transplantation of noncultured and cytokine-cultured cells (54±28% vs. 69±13%). However, the migration of transplanted cells to the bone marrow of other noninjected bones was extremely lower for cytokine-treated cells compared with noncultured cells (2±2% vs. 36±10%). Similar findings were observed for engraftment of CD34+ cells. To enhance the migration of cytokine-cultured cells after IBMT, we similarly transplanted cultured AC133+ cells into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered G-CSF (250 μg/kg/d) for 5 days. Administration of G-CSF stimulated the migration of cytokine-cultured cells to the bone marrow of previously-aspirated right tibia but failed to induce their migration to intact bone marrow of femur. These data indicate that ex vivo manipulation of hematopoietic progenitor/stem cells adversely influences their migration properties to other bone marrow compartments after IBMT. Our data raise caution for future clinical applications of the IBMT method using ex vivo-manipulated hematopoietic stem cells.

Evidence of in vivo osteoinduction in adult rat bone by adeno‐Runx2 intra‐femoral delivery

The bone marrow microenvironment provides a unique opportunity in vivo to assess the role of genes in bone remodeling. The objective of this study was to determine whether Runx2 expression is regulated by rhBMP-2 in vivo and to examine the effect of Runx2 overexpression on bone in vivo. In the in vivo calvaria model we used, rhBMP-2 induced Runx2 protein expression in periosteal cells while in vitro, adenovirus-mediated Runx2 overexpression induced mineralization in mesenchymal stem cells. A single injection of adeno-Runx2 directly into the bone marrow of the right femur in mature rats, and subsequent analysis after 3 weeks, showed a significant bone mineral density (BMD) increase ( approximately 15%) as compared to the controls. The whole-femur mean BMD of the active virus-injected group was 0.193 (g/cm(2)) while that of the control virus-injected group was 0.175 (g/cm(2)) (P < 0.05). In addition, a significant increase (36%) in trabecular BMD at the distal end of the femur was observed. These data demonstrate that directly delivering adeno-Runx2 into bone marrow of adult rats induces osteogenesis and illustrates potential advantages of such approaches over ex vivo gene therapy protocols involving marrow cell isolation, gene transduction, and subsequent in vivo transfer.

2-Methoxyestradiol Suppresses Osteolytic Breast Cancer Tumor Progression In vivo

2-Methoxyestradiol (2ME(2)), a physiologic metabolite of 17beta-estradiol (estrogen), has emerged as a promising cancer therapy because of its potent growth-inhibitory and proapoptotic effects on both endothelial and tumor cells. 2ME(2) also suppresses osteoclast differentiation and induces apoptosis of mature osteoclasts, and has been shown to effectively repress bone loss in an animal model of postmenopausal osteoporosis. Given these observations, we have examined whether 2ME(2) could effectively target metastasis to bone, osteolytic tumors, and soft tissue tumors. A 4T1 murine metastatic breast cancer cell line was generated that stably expressed Far Red fluorescence protein (4T1/Red) to visualize tumor development and metastasis to bone. In an intervention study, 4T1/Red cells were injected into bone marrow of the left femur and the mammary pad. In the latter study, 2ME(2) (10, 25, and 50 mg/kg/d) treatment began on the same day as surgery and was continued for the 16-day duration of study. Tumor cell growth and metastasis to bone were monitored and bone volume was determined by micro-computed tomography. 2ME(2) inhibited tumor growth in soft tissue, metastasis to bone, osteolysis, and tumor growth in bone, with maximum effects at 50 mg/kg/d. Furthermore, tumor-induced osteolysis was significantly reduced in mice receiving 2ME(2). In vitro, 2ME(2) repressed osteoclast number by inducing apoptosis of osteoclast precursors as well as mature osteoclasts. Our data support the conclusion that 2ME(2) could be an important new therapy in the arsenal to fight metastatic breast cancer.