The authors thank Huck Institute&#39;s Flow Cytometry Core Facility and the Histopathology Core Facility of the Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, for providing timely technical support. This work was supported by grants from the American Institute for Cancer Research (KSP), Penn State College of Agricultural Sciences, Penn State Cancer Institute, USDA-NIFA project 4771, Accession number 00000005 to K.S.P. and R.F.P.

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The authors declare no conflict of interest.

These above-described studies provide supportive evidence that the transplantation of Lin- cells is comparable to LSK cells in the generation of 1&#730; murine AML. In addition, the current data also shows that i.p. injection is an efficient and convenient method to establish murine AML compared to intravenous (or r.o.) injection.
In addition to LSK cells, other populations such as granulocyte-monocyte progenitor (GMP), common lymphoid progenitor (CLP), and common myeloid progenitor...

Acute myeloid leukemia (AML) is a type of hematologic malignancy of diverse etiology with poor prognosis1. The generation of AML animal models lays the foundation for the understanding of its complex variations and pathobiology in an effort to discover novel therapies2. Leukemogenesis in mice involves the transplantation of donor cells expressing fusion oncoproteins, including fusions involving the mixed lineage leukemia (MLL) gene to potently induce AML, to mimic the disease in humans3. Various cellular origins of donor cells have been reported in the transplantation of MLL gene-associated AML4, with very little being known about the cells responsible for the disease origin.
Multiple routes have been developed for transplantation in mice; rather than an intra-femoral injection, which directly introduces mutant donor cells into bone marrow5, an intravenous injection that utilizes the venous sinus plexus, tail vein, and jugular vein has been widely used to generate murine AML models6,7,8,9. In the case of retro-orbital (r.o.) injection, various inherent disadvantages, such as volume limitation, high technical demand, few chances for repeated attempts or error, and potential ocular injuries, have been major stumbling blocks with limited or no viable alternatives7. Tail vein injection can have similar problems besides local injuries; to facilitate the procedure, mice often need to be warmed up to dilate their tail veins10. It is also hard to locate the tail vein without an additional light source, particularly in the C57BL/6 strain of mice. For jugular vein injection, research personnel require sufficient training to locate the vein and limit possible complications. In addition, both venous sinus and jugular vein injections need to be performed under anesthesia, which adds another level of complexity. Thus, it is tempting to explore new routes for transplantation to facilitate the establishment of AML murine models.
Intra-peritoneal (i.p.) injection is commonly used to administer drugs, dyes, and anesthetics11,12,13,14,15; it has also been used to introduce hematopoietic cells for ectopic hematopoiesis16 and to transplant bone marrow-derived mesenchymal stem cells in various mouse models17,18,19,20,21. However, it has been infrequently used to establish hematopoietic malignancies in mice, particularly to study AML disease progression.
The present study describes the feasibility of i.p. injection in the generation of AML mouse models, in addition to comparing the transplantation efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor cells. These findings provide a simple and efficient way to generate experimental models of AML and related myeloid leukemias. Such a method has the potential to further our understanding of the disease mechanisms as well as provide a relatively easy model to test experimental therapies.

<table><tbody><tr><td>a-Select competent cells&amp;nbsp;</td><td>Bioline</td><td>BIO-85027</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Sigma Aldrich</td><td>Cat# A-9434</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>Cat# A0797</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V&amp;mdash;Low-Endotoxin Grade</td><td>Gemini bio-products</td><td>Cat# 700-102P</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>GoldBio.com</td><td>Cat# C-861-100</td><td></td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Gibco</td><td>Cat# 11960-044</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline (PBS)</td><td>Corning</td><td>Cat# 21-031-CV</td><td></td></tr><tr><td>EDTA, Disodium Salt (EDTA-2Na), Dihydrate, Molecular Biology Grade</td><td>Calbiochem</td><td>Cat# 324503</td><td></td></tr><tr><td>Fetal Bovine Serum - Premium Select</td><td>Atlanta Biologicals</td><td>Cat# S11550</td><td></td></tr><tr><td>Holo-transferrin, bovine</td><td>Sigma Aldrich</td><td>Cat# T1283</td><td></td></tr><tr><td>Insulin solution human</td><td>Sigma</td><td>Cat# I-9278</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco</td><td>Cat# 12440-053</td><td></td></tr><tr><td>L-glutamine 200 mM (100&amp;times;) solution</td><td>HyClone, Gelifesciences</td><td>Cat# SH30034.01</td><td></td></tr><tr><td>LB broth, Lennox</td><td>NEOGEN</td><td>Cat #: 7290A</td><td></td></tr><tr><td>LB Broth with agar (Miller)</td><td>Sigma Aldrich</td><td>Cat# L-3147</td><td></td></tr><tr><td>Mouse anti-mouse CD45.1 (FITC)</td><td>Miltenyi Biotec</td><td>Cat# 130-124-211</td><td></td></tr><tr><td>Mouse Interleukin-3 (IL-3)</td><td>Gemini bio-products</td><td>Cat# 300-324P</td><td></td></tr><tr><td>Mouse Interleukin-6 (IL-6)</td><td>Gemini bio-products</td><td>Cat# 300-327P</td><td></td></tr><tr><td>Mouse Stem Cell Factor (SCF)</td><td>Gemini bio-products</td><td>Cat# 300-348P</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100x</td><td>Corning</td><td>Cat# 30-002-CI</td><td></td></tr><tr><td>Phenix-Eco (pECO) cells</td><td>ATCC</td><td>CRL-3214</td><td></td></tr><tr><td>Potassium Bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;), Granular</td><td>JT. Baker</td><td>Cat# 2940-01</td><td></td></tr><tr><td>Rat anti-mouse CD117 (c-kit) (APC)</td><td>BioLegend&amp;nbsp;</td><td>Cat # 105812</td><td></td></tr><tr><td>Rat anti-mouse Ly-6A/E (Sca-1) (PE-Cy7)</td><td>BD Pharmingen</td><td>Cat# 558162</td><td></td></tr><tr><td>Recombinant Murine Flt3-Ligand</td><td>Pepro Tech, INC.</td><td>Cat# 250-31L</td><td></td></tr><tr><td>RetroNectin Recombinant Human Fibronectin Fragment</td><td>TaKaRa</td><td>Cat# T100A</td><td></td></tr><tr><td>&lt;em&gt;Trans&lt;/em&gt;IT-293 Reagent</td><td>MirusBio</td><td>Cat# MIR 2705</td><td></td></tr><tr><td>TRI Reagent</td><td>Sigma Aldrich</td><td>Cat# T9424</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco</td><td>Cat # 15250061</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>Cat# 25200-056</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol (BME)</td><td>Sigma Aldrich</td><td>Cat# M3148</td><td></td></tr><tr><td>&lt;strong&gt;Commercial Assays&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit&amp;nbsp;</td><td>StemCell technologies</td><td>Cat# 19856A</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>Cat# 4368813</td><td></td></tr><tr><td>PerfeCTa qPCR SuperMix</td><td>Quanta Bio</td><td>Cat# 95051-500</td><td></td></tr><tr><td>Plasmid Maxi Kit (25)</td><td>Qiagen</td><td>Cat#:12163</td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Ai14&lt;sup&gt;TdTomato&lt;/sup&gt; &lt;/em&gt;mice</td><td>Jackson Laboratory</td><td>Strain # 007914</td><td></td></tr><tr><td>CD45.1 C57BL6/J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Strain # 002014</td><td></td></tr><tr><td>CD45.2 C57BL6/J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Strain # 000664</td><td></td></tr><tr><td>&lt;em&gt;Instruments and Softwares&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe illustrator&amp;nbsp;</td><td>Version 25.2.3</td><td></td><td></td></tr><tr><td>BD accuri C6 flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo 10.8.0</td><td>BD</td><td></td><td></td></tr><tr><td>GeneSys software program&amp;nbsp;</td><td>Version 1.5.7.0</td><td></td><td></td></tr><tr><td>GraphPad Prism version 6&amp;nbsp;</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Hemavet 950FS&amp;nbsp;</td><td>Drew Scientific</td><td></td><td></td></tr><tr><td>7300 Real time PCR system</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Syngene G:BOX Chemi XR5 Chemiluminescence Fluorescence Imaging</td><td>G:Box Chemi</td><td></td><td></td></tr></tbody></table>

intra-peritoneal transplantation for generating acute myeloid leukemia in mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Comparison of the transplantation efficiency of murine AML cells using r.o. and i.p. routes of transplantation 
Previously, the establishment of 1&#730; AML was reported in recipient mice retro-orbitally transplanted with MLL-AF9-transduced LSK cells, and the transplantability of 1&#730; AML cells was demonstrated by serial transplantation30. The present study is the first to evaluate the possibility of using bone marrow Lin- cells to perform transplantation. The p...

Watch this Scientific Journal Video about Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice at JoVE.com

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

intra-peritoneal-transplantation-for-generating-acute-myeloid

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

1.9K Views.  The Pennsylvania State University. Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice. 

Video: Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia 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.

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

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Developmental Biology

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Immunology and Infection

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Medicine

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.

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.

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

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.

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

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Injecting Human HSPCs into the Femur of Sub-Lethally Irradiated Mice

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. 
This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell ...

Biology

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

Source: Krishnan, V. et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/52721">Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent metastasis.&#160;

Source: Krishnan, V. et al. Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. 
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