Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.     

SR-BI in Bone Marrow Derived Cells Protects Mice from Diet Induced Coronary Artery Atherosclerosis and Myocardial Infarction

SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI^+/+apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI^+/+ mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis.     

Direct and Repeatable Bone Marrow Chromosome Preparations from Living Mice

Using a 27 gauge hypodermic needle, bone marrow is aspirated from a lumbar vertebra into 0.1 ml of Hanks' salt solution. The aspirate is kept well mixed in 1% sodium citrate for 15 min, centrifuged, and the cell pellet fixed for 30 min in Clarke's 3:1 ethanol-acetic fixative. After removal of the fixative the cells are suspended in 0.05-0.1 ml of 60% acetic acid, centrifuged and resuspended in 0.03 ml of this fixative. Chromosome preparations are made by spreading the suspension on a slide heated to 60 C.     

Microenvironmental Scenario of the Bone Marrow of Inorganic Arsenic-Exposed Experimental Mice

Exposure to arsenic on a regular basis, mainly through drinking water, agricultural pesticide, and sometimes therapeutic dose, results in various diseases of different tissues including the bone marrow hematopoietic system. Hematopoiesis is a dynamic process by which bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs) generate a relatively constant pool of functionally mature blood cells by the support of microenvironmental components. The present study has been aimed to understand stem cell microenvironmental status during arsenic toxicity and the consequent reflection of dysregulation involving the hematopoietic machinery in experimental mice. Swiss albino mice were experimentally exposed to 10 μg arsenic trioxide/g body weight through oral gavage and 5 μg arsenic trioxide/g body weight intraperitoneally for a period of 30 days. Altered hemogram values in peripheral blood reflected the impaired hematopoiesis which was further validated by the reduced BM cellularity along with the deviated BM cell morphology as observed by scanning electron microscopy post arsenic exposure. The stromal cells were unable to establish a healthy matrix and the sustainability of hematopoietic progenitors was drastically affected in arsenic-exposed mouse groups, as observed in in vitro explant culture. The inability of stromal cells to establish supportive matrix was also explained by the decreased adherent colony formation in treated animals. Furthermore, the flow cytometric characterization of CXCR4⁺ and Sca-1⁺ CD44⁺ receptor expressions confirmed the dysregulation in the hematopoietic microenvironment. Thus, considering the importance of microenvironment in the maintenance of HSPC, it can be concluded that arsenic toxicity causes microenvironmental damage, leading to niche derangement and impaired hematopoiesis.     

Genotoxic and Cytotoxic Effects of Antiretroviral Combinations in Mice Bone Marrow

Commonly used guidelines for the management of human immunodeficiency virus (HIV) infection (highly active antiretroviral therapy, HAART) include drug combinations such as tenofovir disoproxil fumarate (TDF) + lamivudine (3TC) and combivir [zidovudine (AZT) + 3TC] + efavirenz (EFV). These combinations may enhance the genotoxic effects induced by such drugs individually, since the therapy requires lifelong adherence and the drugs have unknown effects during treatment. Thus, the evaluation of the benefits and risks of HAART is of great importance. In order to assess the cytotoxic and genotoxic potential of three concentrations of each of the antiretroviral combinations TDF + 3TC (800 + 400, 1600 + 800, and 3200 + 1600 mg/kg body weight, BW) and combivir + EFV (200 + 100 + 400, 400 + 200 + 800, and 800 + 400 + 1600 mg/kg BW) after two exposure periods (24 h and 48 h), in the present study the in vivo comet assay (single-cell gel electrophoresis) and the mouse bone marrow micronucleus test were used. Neither TDF + 3TC nor combivir + EFV induced DNA damage at any concentrations tested after 24 h or 48 h using the comet assay. After 24 h, both combinations increased the micronucleus frequency at all concentrations tested. After 48 h, combivir + EFV increased the micronucleated polychromatic erythrocyte (MNPCE) frequency at the two highest concentrations tested. Polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE) ratio was high for both combinations, suggesting that they can be mitogenic. Since genotoxicity may be related to carcinogenesis, it is necessary to conduct further studies to verify the long-term mutagenic effects of these drugs.     

Smad3基因剔除的骨髓细胞移植对小鼠的影响

目的通过小鼠骨髓细胞剔除Smad3基因,观察小鼠病理变化以及免疫T细胞状态。方法将Smad3基因剔除Smad3-/-)的小鼠骨髓细胞和野生型(Smad3+/+)小鼠骨髓细胞分别移植给60Co射线照射GFP小鼠。观察骨髓移植后GFP小鼠体征变化,第6周处死小鼠,取肠道固定,HE染色观察其病理变化,流式细胞技术检测淋巴结中T细胞变化。结果移植Smad3-/-骨髓细胞的GFP小鼠逐渐消瘦,大肠出现炎症;淋巴结中活化型的CD4+CD62LloT细胞增多。结论骨髓细胞TGF-β信号受阻,可导致小鼠患炎症疾病,引起免疫T细胞活化。     

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism >80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.     

四物汤对再生障碍性贫血小鼠骨髓细胞增殖的影响研究

目的研究四物汤对再生障碍性贫血（AA）小鼠骨髓细胞体外增殖的影响,探讨其治疗AA的机制。方法采用流式细胞仪、骨髓造血祖细胞培养等技术,检测四物汤对AA小鼠骨髓细胞的增殖变化。结果 四物汤能促进骨髓有核细胞进入G2／S期、增加CFU—GM、CFU-E、BFU-E集落数,且与自然恢复组有明显增强的差异。结论 四物汤在体内有促进AA小鼠骨髓细胞增殖的作用,为补血药治疗AA提供了实验与理论依据。     

Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.     

Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen

JC virus (JCV), a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML). In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs) isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases and the opportunities for the use of this model in development of therapeutic strategies.     

载脂蛋白E基因敲除小鼠用于动脉粥样硬化研究的进展

载脂蛋白（apolipoprotein,ApoE）基因敲除小鼠是目前研究动脉粥样硬化发生发展机制的最为理想的动物模型之一,尤其是近年来,又成为易损斑块动物模型研究的热点。有关apoE^-I-小鼠动脉粥样硬化斑块病理特点、炎症在斑块破裂中的作用及对其干预治疗等研究,近来又有了许多新的发现。     

Allergen-Induced Bone Marrow Eosinophilopoiesis and Airways Eosinophilic Inflammation in Leptin-Deficient ob/ob Mice

Asthma and obesity are growing epidemics in the world. It is well established that obesity worsens the asthma outcomes. High-fat diet-induced obesity in mice exacerbates the pulmonary eosinophilic inflammation. We have used wild-type (WT) and ob/ob mice to further explore the mechanisms by which obesity aggravates the pulmonary eosinophilic inflammation. The eosinophil (EO) number in bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow were evaluated at 24, 48, and 72 h after ovalbumin (OVA) challenge in sensitized mice. The basal EO number (phosphate-buffered saline (PBS)-instilled mice) in lung tissue was about 3.5-fold greater in ob/ob compared with WT mice. OVA challenge in ob/ob mice promoted an EO accumulation into the lung that was accompanied by a lower emigration to airways lumen (BAL fluid) in comparison with WT mice. OVA challenge also markedly elevated the number of mature and immature EO in bone marrow of ob/ob mice at 24 h compared with WT group. Blood EO at 48 h was markedly greater in ob/ob mice. Tumor necrosis factor (TNF)-α and interleukin (IL)-10 levels in BAL fluid were significantly higher in ob/ob mice, whereas no changes for IL-5 and eotaxin were found. The IL-6 levels were significantly lower in ob/ob mice. In conclusion, OVA challenge in ob/ob obese mice potentiates eosinophilopoiesis and promotes an accumulation of EO into the lung tissue, delaying their transit to airways lumen. The longer EO remain into the lung tissue is likely to contribute, at least in part, to the asthma worsened by obesity.     

GTW对小鼠骨髓细胞微核和精子畸形的影响

研究了雷公藤多甙(GTW)的抗生育剂量、中间剂量和治疗剂量对昆明种小鼠骨髓细胞微核和精子畸形的影响,并对用药后其体重变化和睾丸/体重的比例进行比较。骨髓微核试验和睾丸/体重比变化分析均显示中间剂量组和治疗剂量组有不同程度的遗传毒性,且存在剂量-效应关系,但小于阳性对照;精子畸形率呈下降趋势,生育力的可复性可能也是下降的;体重变化可见中间剂量组存在较高的代偿能力。     

小鼠骨髓间充质干细胞的培养、鉴定和Survivin的表达研究

为培养及鉴定小鼠来源骨髓间充质干细胞,并测定细胞中Survivin的表达情况,采用全骨髓培养法获取骨髓间充质干细胞,绘制生长曲线,流式细胞仪检测细胞表面标志物,行成骨、成脂检测,RT-PCR测定Survivin表达情况.结果表明培养出的细胞呈长梭状成纤维细胞样,经流式细胞仪检测细胞表面高表达CD29、CD34、CD44、SCA-1,低表达CD117;细胞曲线显示传代细胞培养1～3d生长缓慢,第4d生长加快并于第7d达到高峰;成骨诱导20d经茜素红染色呈红色结节,成脂诱导14d油红O染色显示有大量脂质沉淀;RT-PCR结果显示Survivin mRNA阳性表达.经全骨髓培养法可以培养出大量骨髓间充质干细胞,同时Survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程.     

Preparation of Bone Marrow Sections

It is possible to prepare in the following manner sections of aspirated bone marrow suitable for staining by the majority of conventional methods. The aspirated marrow is ejected into a small test tube containing 0.5 mg heparin powder. At any convenient time during the next hour the material is poured into a watch glass, and the individual marrow particles, free from excess blood, transferred by means of a thin pointed plastic rod to a jar containing 10-15 ml of fixative. Any of the commonly employed fixatives may be used. After not less than 1 hr, the marrow particles are poured onto filter paper from which they are removed to a test tube containing 70% ethanol. During dehydration with absolute ethanol, clearing with two changes of chloroform and embedding in paraffin wax, the particles remain in the tube. After cooling, the tube is broken and the material, found at the apex of the round-ended block, is readily accessible for cutting. Concentration is sufficient to allow the whole sample to be studied in a small number of serial sections. Experience has shown that these sections are equally satisfactory for the study of morphology, cytology, or mineral content.