This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

注:伦理学声明:此协议已审查和批准由西蒙·弗雷泽大学的动物护理委员会（UACC;允许数字1037K-12和1060K-03），并符合加拿大议会动物保育，美国国立卫生研究院指南护理和使用实验动物，以及EEC理事会指令。 1.特别注意事项注:白消安是细胞毒性。处理并根据材料安全数据表和制度规定进行处置。 <ol><li>执行所有的技术在无菌层流罩。 </li><li>消毒通过包装在合适的包装材料，诸如剥离封装，并高压灭菌，使用前的仪器。 </li><li>而相比于照射为感染的风险是低带马利兰调节，仅在层流罩的前2周后移植处理的动物。 </li></ol> 2.调理受体小鼠<ol><li>稀马利兰至3毫克/毫升，用无菌磷酸盐缓冲盐水（PBS）中。 注意:为了让新鲜稀释至白消安注射液使用的每一天之前是很重要的。 </li><li>日常管理20毫克/公斤的白消安稀释到受体小鼠通过IP注射。 </li><li>重复的20毫克/千克马利兰每日IP注射，直到60-100毫克总剂量/千克已经交付（ 即，对于一个80毫克/公斤的总剂量，给药白消安，连续4天的20毫克/千克）。 </li></ol> 3.隔离供体骨髓细胞的注:此协议已被成功地用于从最多5供体小鼠中分离和制备BM细胞。通常每只小鼠的细胞产量是约30-40百万DC培养上清，这足以移植12-16受体小鼠。如果需要更多的供体小鼠中的协议可能需要作相应调整。 <ol><li>继白消安空调euth的最后一天anize使用的CO 2与GFP供体小鼠（1至6个月大）（或通过在接纳机构其他安乐死过程）。为了避免移植并发症使用同基因供体是同性的收件人。 </li><li>喷小鼠用70％的乙醇。升降机皮肤在腹部，并使用外科剪刀通过从腹腔向上朝向脚踝腿部皮肤做一个切口。握住脚，坚决从拉向臀部露出腿部组织脚踝的皮肤。 </li><li>修剪掉肌肉和脂肪组织从股骨以暴露髋关节。 </li><li>同时轻轻拉动脚伸腿，按压髋关节的剪刀。剪刚股骨，注意不要切股骨本身的头上。 </li><li>为了帮助保持无菌，由脚抱腿和摩擦骨表面灭菌组织清理从骨子里任何剩余的组织。 </li><li>通过在膝关节切削分离股骨和胫骨和放置在含有无菌PBS培养皿股骨。在冰上孵育。 </li><li>取出并削减在那里的腓骨连接到胫骨点丢弃腓骨。将胫骨在培养皿与股骨和在冰上孵育。 </li><li>对于另一条腿重复步骤3.2-3.7，并在必要时，附加的供体小鼠。下面除去的骨头，消毒的外科手术工具用热珠灭菌或使用一组新的无菌工具用于随后的步骤。 </li><li>对于股骨，抱钳股骨，用手术剪刀仔细地&#39;刮&#39;的远端脱骨。去除尽可能少，以暴露骨髓腔​​必要的骨。 </li><li>填充用3毫升无菌PBS的注射器，并附加一个23政针。仔细孔针插入BM腔冲洗BM到无菌培养皿。一定要凑髓腔用针点，以保证除去所有需要的细胞。以下提取，确保红色BM不再可见和骨现在显示为白色。 </li><li>随后的股骨重复步骤3.9-3.10，汇集所有的BM在同一培养皿。 </li><li>为胫骨，保持胫骨钳，并仔细&#39;剃须&#39;，其中所述胫骨附着到膝盖以暴露骨髓腔​​的末端。使沿其中可见红色BM结束骨第二晋级。 </li><li>填用3ml无菌PBS的注射器，并附加一个地下25针。轻轻插入针头插入骨髓腔（从附着到膝盖该端），并刷新BM到从股骨含有BM培养皿。刮去骨髓腔的壁与针以除去所有的细胞。以下提取，确保红色BM不再可见和骨现在显示为白色。 </li><li>随后的胫骨，重复步骤3.12-3.13，汇集所有的BM在同一个培养皿。 </li><li>轻轻磨碎的BM与1毫升枪头分离的CELLS。 </li><li>通过40μM过滤器篮的细胞悬浮到无菌50mL离心管中。冲洗用PBS菜获取任何剩余的细胞，并通过过滤器转移到这一点的离心管中。 </li><li>离心5分钟，在450×g离心在4℃下。取出并弃去上清液。 </li><li>重悬在3ml红细胞裂解缓冲液沉淀。在冰上孵育8.5分钟。 </li><li>加约30毫升无菌PBS中以猝灭裂解缓冲液中。 </li><li>离心5分钟，在450×g离心在4℃下。取出并弃去上清液。 </li><li>重新悬浮于1ml无菌PBS的沉淀。保持在冰上。 </li><li>取一小等分试样的细胞匀浆和稀释用PBS（1:100 1小鼠; 1:200为2小鼠等）使用血细胞计数器来量化细胞数。 </li><li>稀释细胞悬液，以8×10 6个细胞/ ml，并在冰上孵育直至注射。 </li></ol> 4.骨髓移植<ol><li>网络连接LL 0.5ml注射器（用28克胰岛素针）用300微升稀释的细胞悬浮液对每个小鼠进行注射。一定要消除存在于注射器任何气泡。 </li><li>上放置装有一个加热垫的空调受体小鼠鼠笼并允许尾静脉扩张。 </li><li>就拿收件人鼠标和地方约束装置。擦拭用外科纱布浸透70％乙醇的尾部。 </li><li>牢固地保持在尾部，并与斜面向上，轻轻插入针头插入的侧面尾静脉中的一个，并注入该细胞悬浮液。 </li><li>保持手术纱布在注射部位，直到前引入鼠标到一个新的干净的笼子出血停止。 </li></ol> 5.估计BM嵌合体的程度注:嵌合体的水平是在外周血典型变量1周后的骨髓移植，因此嵌合体的水平，通常估计在至少2周后的骨髓移植。詹水平erism应达到&gt; 80％的GFP +细胞在外周血3-4周内后骨髓移植。 <ol><li>制备FACS缓冲液（2mM EDTA的+ 2％胎牛血清的PBS血清）。 注:FACS缓冲液可以储存在4℃几周。 </li><li>鼠标放置在一个约束管刮胡子的皮毛上的小腿后侧暴露侧隐静脉。 </li><li>薄外套凡士林和皮尔斯隐静脉将25g针头的腿。 </li><li>收集大约50微升血液与肝素涂层的毛细管。加血液至含有1ml的FACS缓冲液中的微离心管中。混合管通过倒置并储存在冰上。 </li><li>按住注射部位的手术纱布，直到出血停止返回鼠标回到笼子前。 </li><li>离心血液样品5分钟，在900×g下。取出并弃去上清液。 </li><li>重悬在500μl的红细胞裂解缓冲液沉淀。在冰上孵育8.5分钟。 </li><li>加入1毫升的FACS缓冲液，淬灭裂解缓冲液中。 </li><li>离心样品5分钟，在900×g下。取出并弃去上清液。 </li><li>执行在500μl的红细胞裂解缓冲液的第二裂解步骤。在冰上孵育8.5分钟。 </li><li>加入1毫升的FACS缓冲液，淬灭裂解缓冲液中。 </li><li>离心样品5分钟，在900×g下。取出并弃去上清液。检查球现在是白色的。 注意:如果该粒料仍然出现红色，附加的裂解步骤可能需要在进行下一步骤之前。 </li><li>重新悬浮于1ml FACS缓冲液沉淀。离心样品5分钟，在900×g下。取出并弃去上清液。 </li><li>重悬在300微升FACS缓冲液将沉淀，并使用流式细胞术定量的GFP +细胞（注意:在这一点上可进行该细胞的任何期望的免疫染色，使用标准技术，前流式细胞仪分析）。 </li></ol>

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The authors have nothing to disclose.

马利兰调理允许高层骨髓嵌合小鼠造血细胞是容易检测的产生。可以无需照射设施来执行此调节。此外，骨髓抑制与剂量在这个协议中概述马利兰的耐受性良好，最小化毒副作用引起的清髓性剂量的辐射，并因此可能是一个更合适的技术，以产生BM嵌合在年轻的，年老，或患病的小鼠更容易清髓性照射。一些RIC协议使用低的，非myleoablative剂量全身照射，以尽量减少不良副作用，但这些协议仍然需?...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

<table><tbody><tr><td>Busulfex</td><td>Otsuka Canada Pharmaceutical Inc.</td><td>DIN 02240602</td><td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td></tr><tr><td>ACK Lysing Buffer</td><td>Life Technologies</td><td>A10492-01</td><td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>12483-020</td><td></td></tr></tbody></table>

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&nbsp;>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.

60-100毫克/公斤白消安是普遍受到老鼠的耐受性。然而，动物通常在调节阶段（〜5-10％），因此饮食减肥可能需要与对待诸如照射葵花籽鼓励饮食进行补充。 &gt; 80％的GFP +细胞在外周血嵌合和BM的水平应始终实现使用60-100毫克/公斤马利兰7。我们的实验室已成功地利用这个协议产生超过100嵌合体小鼠&gt; 80％GFP +细胞在外周血和骨髓，几乎我们所有的BMTs使用同基因捐献者和接受者的时候是?...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

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

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

白消安作为骨髓抑制剂在小鼠生成稳定的高水平骨髓嵌合体

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

busulfan-as-myelosuppressive-agent-for-generating-stable-high-level

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.2K 次观看.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

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

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

骨髓增殖性疾病的转导移植小鼠模型

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

科研

癌症研究

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

医学

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

发育生物学

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

使用急性淋巴细胞白血病患者来源的异种移植小鼠模型评估嵌合抗原受体T细胞相关毒性

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

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.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

免疫与感染

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

白消安作为骨髓抑制剂在小鼠生成稳定的高水平骨髓嵌合体

摘要

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此视频中的章节

骨髓增殖性疾病的转导移植小鼠模型

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

应用造血干细胞移植评估骨髓增生异常综合征的起源

使用急性淋巴细胞白血病患者来源的异种移植小鼠模型评估嵌合抗原受体T细胞相关毒性

股骨骨髓的愿望在活体小鼠

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

小鼠骨髓移植程序研究克隆造血症

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法