We thank all the members of the Majeti lab for their help, support, encouragement, and inspiration over the years. We acknowledge the Flow Cytometry Core of the Stanford Stem Cell Institute, the Binns Program for Cord Blood Research, and the patients for donating their samples. For human samples, normal donor human bone marrow and peripheral blood cells were obtained fresh from AllCells or the Stanford Blood Center. We thank the Nakauchi lab at Stanford University for donating the pBac-AkaLuc-tdTomato plasmid. Above all, we would like to thank the veterinarians and animal control staff at the Veterinary Service Center at Stanford who take care of our mice. In particular, Mike Alvares, supervisor of the animal center, has been so thorough in his management of the mice that it is no exaggeration to say that without him, our research would not have been possible.
This work was supported by NIH grants 1R01HL142637 and 1R01CA251331, the Stanford Ludwig Center for Cancer Stem Cell Research and Medicine, and the Blood Cancer Discoveries Grant program through The Leukemia &#38; Lymphoma Society, The Mark Foundation for Cancer Research, and The Paul G. Allen Frontiers Group, all to R.M. R.M. is a recipient of a Leukemia and Lymphoma Society Scholar Award. Y.N. was supported by the Nakayama Foundation for Human Science and a Stanford University School of Medicine Dean&#39;s Postdoctoral Fellowship. A.E. was supported by the NCI under award F32CA250304, the Advanced Residency Training Program at Stanford, and the American Society of Hematology Scholar Award.

Six- to ten-week-old male or female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used in this protocol, but it can be applied to all types of mice. The irradiation depends on the experimental content and the type of mice, whether or not to irradiate the mice depends on the research objectives. All animal procedures described here were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by Stanford University&#39;s Administrative Panel on Lab Animal Care (APLAC #22264). All normal blood cell populations were sorted out fresh. Human AML samples were obtained from patients at the Stanford Medical Center with informed consent, according to institutional review board (IRB)-approved protocols (Stanford IRB, 33818).
1. Intrafemoral injection of cell lines, hematopoietic stem and progenitor cells (HSPCs), or leukemic stem cells (LSCs)
NOTE: To easily analyze the injected cells with an imaging device, an Akaluc/tdTomato-positive K562 cell line was generated, and AkaLumine-HCl/Akaluc was used as a substrate10. If imaging equipment is not available, cells can be labeled with a fluorescent dye by lentivirus or PiggyBac and analyzed by flow cytometry. Regardless of the details of cell preparation, having a way to prove that the cells administered into the bone marrow have reliably entered the BM is important for improving this technique (Figure 1).
<ol>
	<li>Prepare cell suspensions (up to 3 &#215; 106 blood cells) with 20 &#181;L of fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS) + 2 mM EDTA) or Thaw medium (IMDM + 20% FBS + Pen/Strep) in PCR or 1.5 mL tubes and keep on ice before injection. 
	NOTE: Avoid making air bubbles while suspending the cells with the needle. Bubbles should be removed as much as possible right before the injection, as injecting air bubbles can cause sudden death of mice.</li>
	<li>Condition 6-10-week-old adult mice with 200 cGy (225 kV, 13.3 mA, total 2 Gy [Dose control mode]) up to 48 h prior to injection of the cells.</li>
	<li>First, anesthetize the mice with isoflurane inhalation in an induction chamber and maintain at 2% isoflurane in 100% oxygen at a flow rate of 1-2 L/min via a nose cone to reach a steady state of anesthesia. Check the depth of anesthesia with a toe pinch reflex and slow, steady breathing, and maintain this state throughout the isoflurane procedure. 
	NOTE: Monitor the depth of the anesthesia every 3-5 min throughout the procedure to ensure there is no change in heart and respiratory rates with surgical manipulation and/or ear, toe, and tail pinch. The color of mucous membranes and skin can be used as an indicator of oxygenation, as they will be pinkish if the mice are in good condition. Exercise caution when opaque drapes are placed over the mouse, as it is not always possible to see what is happening with the mouse.</li>
	<li>Prior to the procedure, administer mice with 10 mg/kg carprofen subcutaneously to minimize pain and warm 0.5-1.0 mL of 0.9% saline for supportive care.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse to avoid corneal drying.</li>
	<li>Keep the mouse on a heat pad or other thermostatically controlled surface to prevent hypothermia during the procedure. 
	NOTE: After the procedure, a temperature-adjustable heating pad is used. The body temperature of the mouse is typically 37-39 &#176;C, and the pad should ideally be slightly lower than this. In practice, the temperature is often set at 32-36 &#176;C.</li>
	<li>Disinfect the entire leg containing the femur to be injected with three sets of alternating scrubs (alternating with either a povidone-iodine or a chlorhexidine scrub and 70% ethanol-soaked gauze sponges). 
	NOTE: 1) If the operator is left-handed, the left femur of the mouse might be easier to inject than the right femur. 2) In this study, the fur was not removed for injections to avoid unnecessary skin damage, and the needle entry site can be easily located without cutting the fur off. However, in mice with dark fur, it may be challenging to locate the puncture site, in which case, consider cutting off the fur as it is quick and easy to start the injection.</li>
	<li>Pinch the femur gently with the thumb and index fingers to stabilize the leg. Push the tibia with either the ring or the fifth finger to keep the tibia bent from the femur. Positioning is important for successful injection (Supplementary Figure S1-S5). 
	NOTE: Use forceps to pinch the bone to prevent injury to the fingers; however, it might be difficult to feel the shaft of the femur and will add time to the procedure.</li>
	<li>Insert an empty sterile 27 G needle (1/2 inch) with an attached syringe just under the patellar tendon so that the needle is lodged securely between the two condyles of the femur. Use the edge of the needle and peck the patellar tendon on top of the femur lightly a couple of times to find the best place to insert the needle (Supplementary Figure S1-S5). 
	NOTE: A knowledge of the anatomy of the mouse knee area is necessary before performing this procedure. Beginners should take the time to understand the anatomy of the area and practice the procedure on an euthanized mouse before attempting it on a live mouse.</li>
	<li>Swivel the needle outward and upward to ensure it is parallel with the shaft of the femur. 
	NOTE: This maneuver provides a reliable path to the marrow cavity, facilitates retrieval of marrow contents from the femoral shaft, and minimizes discomfort to the animal from the procedure.</li>
	<li>Turn the needle in circles while slowly advancing into the femoral marrow cavity. Insert the needle until there is a noticeable reduction in resistance. Confirm the correct positioning of the needle by gently moving the syringe laterally. If it is possible to touch the edge of the needle with the fingers coming outside the bone, go back to step 1.9 and find another angle and place to drill the needle down.
	<ol>
		<li>Ensure that the needle&#39;s entry angle is perpendicular to the bone end-face. 
		NOTE: Theoretically, the needle must be injected parallel to the femur and must be at a 90&#176; angle to the bone ends. If there is resistance from the interior surface, the needle has been correctly placed in the femoral cavity. To make sure the needle is parallel to the bone, place a light on the side of the bone and observe the shadow of the bone parallel to the shaft of the needle. The decrease in resistance upon entry into the bone marrow cavity depends on the age of the mouse: the younger the mouse, the easier it is to recognize the decrease in resistance.</li>
	</ol>
	</li>
	<li>Create negative pressure by gently pulling the needle plunger back while moving the needle back and forth within the BM cavity. If the needle is in the BM cavity, some blood/marrow will come out in the syringe.
	<ol>
		<li>If the blood/marrow is not seen, change to a new needle and try to find the same route. 
		NOTE: The reason for changing to a new needle is that once the bone clogs the needle, it becomes difficult to check for the backflow of the blood/marrow. Minimizing the number of injection routes into the BM cavity is crucial, as cells injected into the bone marrow have the potential to leak out of the femur through previous entry points. If multiple injection routes are necessary, leaking risk can be reduced by injecting cells gradually rather than a quick bolus.</li>
	</ol>
	</li>
	<li>Remove the needle slowly and insert the cell-containing syringe through the same route. Try to insert the needle until it stops (at the edge of the BM cavity) and pull back a little bit (1-2 mm) from there to inject the cells easily. Prior to depressing the plunger and releasing the cells, aspirate slightly and check for blood/marrow to ensure the correct placement of the needle. Check the backflow of the blood/marrow first and then push the syringe slowly to inject the cells. 
	&#8203;NOTES: It is very important to remember the route and angle of the first injection when switching to a new needle. If the same route cannot be found, try again with the original needle used to make the first route. A new needle may be used at this time, but if the original needle is used, the jammed bone should be pushed out once before use. Do not use the needle with the cells to find a new route to inject; otherwise, pieces of bone stuck in the needle may prevent cell dispersal. Although the BM cavity is very small, the injection volume should be less than 30 &#181;L, considering the dead cavity.
	<ol>
		<li>Ensure that the speed of injection is slow to prevent air in the syringe from entering the bone marrow and to prevent cells from leaking from the puncture site. If resistance is felt while pushing the syringe, move the needle up and down to find a less resistant area in the cavity.</li>
	</ol>
	</li>
	<li>Once the cells are successfully injected into the femur, remove the needle and syringe from the mouse while maintaining pressure on the syringe. 
	NOTE: Try again with the other femur if the injection cannot be completed. The procedure is very stressful for the mouse, even under anesthesia. Always monitor the vital signs (heart and breathing rate) of the mouse and try to finish the procedure as soon as possible.</li>
	<li>Remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding. During the recovery, keep the mouse on a heating pad or other thermostatically controlled surface. Monitor all mice to verify recovery from anesthesia prior to placing them back on the mouse rack. 
	NOTE: There should be no complication or distress post aspiration if done properly. The procedure will be completed when anesthetized mice have recovered and are able to ambulate and reach food and water.</li>
	<li>Observe the mice for signs of distress or infection post-procedure over the next 24 h. Signs of distress or infection include constant bleeding, anemia, and lethargy. If any of these signs are seen post-procedure, euthanize the animal(s) by CO2 inhalation or cervical dislocation according to the animal handling protocol.</li>
</ol>
2. Aspiration of bone marrow cells from the femur for the FACS analysis
NOTE: The procedure for aspirating the BM cells from the mice is very similar to the methods described in section 1 and can be learned from some previous literature9,11,12. The following is an overview of some differences between the BM injection and aspiration protocols.
<ol>
	<li>Prepare 500 &#181;L of PBS + 10 mM EDTA (cell suspension medium: CSM) per sample for suspending the BM aspiration.</li>
	<li>Wet a 0.5 mL 27 G syringe with CSM before aspirating the BM. Fill the syringe with 200-500 &#181;L of CSM and immediately expel it. Repeat this procedure 2-3x.</li>
	<li>Aspirate the BM gently by withdrawing the plunger on the syringe yielding 20-50 &#181;L of mouse BM. Then, fully remove the needle from the femur and suspend the aspirated sample in CSM (500 &#181;L in a 1.5 mL tube) for the following staining step. Remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding during recovery. Monitor all mice to verify recovery from anesthesia prior to placing them back on the mouse rack. 
	NOTE: BM aspiration/sampling can be repeated, but the repeat procedure on the same day must be performed on the opposite femur to prevent repeated trauma to the same leg. Although there is little information on the frequency of BM aspiration, we believe that femoral BM aspiration is generally repeated every 4 weeks and can be performed at least 3-4x in total without any issues (e.g., infection) based on our experience. However, it should be kept in mind that the bone marrow on the contralateral side of the transplanted bone marrow generally has lower cellular engraftment.</li>
	<li>Pellet cells from the bone marrow aspirate by a 5 min spin at 300 &#215; g, 4 &#176;C.</li>
	<li>Aspirate the supernatant, add 0.5-1 mL of ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) to each tube, and vortex to resuspend the cells. Place on ice for 5-10 min.</li>
	<li>Filter each tube through nylon mesh into a new tube.</li>
	<li>Rinse the tube with 1 mL of FACS buffer and add it through the nylon mesh into a new tube as well.</li>
	<li>Pellet cells by a 5 min spin at 300 &#215; g, 4 &#176;C.</li>
	<li>Aspirate the supernatant and add the staining solution containing the desired antibodies13. Place on ice for 20 min.</li>
	<li>Wash the cells and analyze them as per the experimental setup.
	<ol>
		<li>Wash cells with FACS buffer (PBS, 2% FBS, 2 mM EDTA) and stain them with antibodies for 30 min on ice in a total volume of 50 &#181;L. Wash and stain the cells with propidium iodide (PI) at a final concentration of 1 &#181;g/mL immediately before the analysis or sorting. Perform post sort analyses to verify the purity of the sorted cell populations. 
		NOTE: All antibodies used for flow cytometry are detailed in the Table of Materials.</li>
		<li>Use the following FACS gating strategies (Figure 2-7) to analyze the engrafted cells from the mouse bone marrow.
		<ol>
			<li>Distinguish populations of cells by their forward (size) and side scatter (granularity) properties.</li>
			<li>Perform doublet discrimination by plotting FSC-H vs FSC-W, following SSC-H vs SSC-W.</li>
			<li>Exclude dead cells.</li>
			<li>Distinguish populations of cells by human CD45 and mouse CD45. These antibody staining combinations enable the detection of human CD3, CD19, and CD33 as well within the human CD45 fraction. 
			NOTE: Before staining the samples, remember to use mouse and human Fc blocks to prevent non-specific antibody binding. We usually stain with the Fc blocks (mouse + human) for 10 min on ice and then add the antibodies without washing the cells. Refer to each company&#39;s instructions on how to use the Fc block antibodies.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Intrafemoral Injection for Bone Marrow Study in Live Mice

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R.M. is on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, and 858 Therapeutics and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. R.M. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics.

Murine xenograft models are important for studying both normal and pathological human hematopoiesis. The BM is the source of hematopoiesis3; therefore, studying hematological diseases requires the successful engraftment of rare human stem cells into the murine BM. So far, transplantation methods such as a tail vein, retroorbital vein, and IF injection have been reported, and it is known that any of these methods can engraft transplanted cells. IF injection is a transplantation method that has been used to ensure the implantation of a very limited number of human-derived cells into immunodeficient mice, and its engraftment rate is very stable compared to other methods6,7,8,9,11. In addition, the utility of direct intrabone marrow transplantation has already been reported in clinical practice5,19.
This procedure can be easily performed at any facility with an anesthesia machine. Regarding the time of the procedure, it is better to get it done as quickly as possible, both to reduce the burden on the mice and for the cells to be harvested. This procedure can be performed in one or both legs, allowing for engraftment and bone marrow analysis of either or both legs. However, because of the strain on the mice, we usually collected from only one leg.
Stabilizing the leg to ensure that the penetration of the bone is possible without movement of the patella and to guarantee finding the same hole a second time when the first needle gets removed is the most critical point to injecting the cells successfully. First, the mouse leg should be placed firmly on the ground, and the femur should be held firmly by both the index finger and thumb. In this way, the area near the patella is immobilized and stabilized. More importantly, the palm holding the femur should be as stable as possible on the ground. In doing this, it is important not to choke the mouse by pushing on it with the hand. Second, once the mouse is secured correctly, the secret to success is to create the entry path with the first needle, keeping an eye on the needle as it is removed, and then insert the second needle. It is advisable to keep the syringe containing the cells in a place that is readily accessible without taking one&#39;s eyes off the mouse&#39;s lap.
Still, the following points need to be considered when performing an IF injection. Future studies should focus on understanding the potential effects on the structure of the BM or on the type and number of cells that can be harvested from the BM after IF injection.
In rare cases, the bone at the puncture site thickens, and subsequent cell aspiration becomes difficult after 2-3 months of injection. In this case, the BM can be harvested from the non-injected leg. However, the engraftment rate on the opposite femur is generally lower than at the transplanted site6. From our experience, the maximum number of cells that can be safely injected into the BM of a mouse is 5 &#215; 106 cells, and more than that may cause sudden death of the mouse due to symptoms of cellular embolism. Only one case of sudden death was experienced with IF injection of 1 &#215; 107 patient cells, but when the diluent was increased from 20 to 50 &#181;L, and the cells were injected very slowly, there was no issue. However, since there are reports on the capacity of BM20, it is necessary to optimize the number of cells, the amount of suspension medium, and the engraftment to retain the cells in the BM efficiently.
Based on our experience, we believe that the transplantation efficiency of IF injection is at the same level as the efficiency of intravenous transplantation into newborn mice. However, irradiating newborn mice is expected to cause many side effects, such as growth disturbance and early death, which may significantly impact the experimental system. Therefore, we believe that IF injection is an important implantation method for experimental systems that require long-term follow-up of mice. However, it should be understood that there is a discrepancy in cell engraftment in both peripheral blood, bone marrow on the side of the transplant, bone marrow on the opposite side of the transplant, and samples obtained by euthanizing and crushing all bones (typically the sternum, pelvis, femur, tibia, fibula, and spine).
This protocol for BM procedures in mice involves a two-step process that requires perforation of the cortical bone with a first needle followed by cannulation of the orifice with a needle of the same gauge for cell injection.
This step-by-step procedure takes less than 30 s for an experienced researcher if everything is ready to set up. The success rate is extremely close to 100%, although this must be confirmed by assessing engraftment. As described in this paper, if an imaging system can be built to track the cells, the cells can be transplanted and immediately tracked to see how they behave in the body.
If the described protocol is followed, the chances of finding the same hole with the second syringe are close to 100%. If the same spot is found, the second syringe needle can be inserted with little or no resistance felt. If the needle is inserted without resistance, always check for reverse bleeding to confirm that the needle has been inserted into the bone marrow. With experience, it becomes possible to know that the needle has been inserted into the bone marrow without confirming reverse blood flow or feeling a decrease in resistance during needle insertion. To expertise the technique, it is important to use IACUC-approved practice mice under anesthesia or already dead mice before the actual experiments.
There is a report using a pediatric spinal tap needle to efficiently and conveniently inject and sample cells in mice BM with one perforation of the bone12. Although this procedure has the advantage of not requiring a needle change, it is difficult to feel using the fingers whether the needle gets into the BM cavity, and it takes time to complete the procedure overall. Further, generally, these instruments cost more than the standard disposable needle used in this protocol.
IF injections provide an efficient disease model using valuable human samples and contribute significantly to the elucidation of disease states. Seeing is believing, and we believe that the detailed protocols presented in this video are important for the widespread adoption of IF injections or aspirations, which were thought to require specialized skills. We hope that this technique will be more widely used and that the pathophysiology of many diseases will be further elucidated.
For the continuous bone marrow analysis by intrafemoral aspiration, it has been previously reported that it is possible to continuously analyze cells in the BM of mice transplanted with HSPCs intravenously9. However, in this study, we have shown that it is possible to perform IF injection of various types of human-derived cells into immunodeficient mice, which are generally susceptible to stress, and to analyze the cells in the BM (at least every 4 weeks for three times) safely and repeatedly while the mice are still alive.
With respect to flow cytometric staining, before staining the samples, mouse and human Fc blocks must be used to prevent non-specific antibody binding. Fc receptors are found on B cells, monocytes, macrophages, and dendritic cells. They bind antibodies via their constant Fc domain rather than the antigen-specific Fab domain. Such nonspecific reactions of antibodies may lead to false-positive data, which could make interpretation of data difficult.
In summary, we have developed a protocol for successful IF injections using various types of human cells and serial bone marrow analysis of live immunodeficient mice. We do hope that many researchers will use this technique to help elucidate the pathophysiology of various diseases.

The blood system is maintained throughout life by hematopoietic stem cells (HSCs), which reside in bone marrow (BM)1,2. To study dynamic changes in the BM environment, it is important to understand the biology of both normal and malignant hematopoiesis3,4. Transplantation of HSCs directly into human BM yields higher engraftment than peripheral blood (PB) infusion, but high procedural complexity and increased risk of infection preclude this method from being part of standard practice5. In mice, procedures that facilitate BM access without sacrificing the animals provide a resource to serially monitor hematopoiesis. The described procedure aims to produce mice with high engraftment of transplanted cells and allow for serial sampling of the BM of live mice. This paper will focus on producing xenograft models using immunodeficient mice engrafted with human cells, which are more challenging to produce than mouse-mouse allotransplantation models. Compared to conventional transplantation of hematopoietic stem and progenitor cells (HSPCs) through the tail or retroorbital vein, the advantages of this procedure are high cellular engraftment with a low amount of starting material.
Although intrafemoral (IF) cellular injection into the BM cavity of mice is commonly used to study human HSPCs in vivo6,7,8, a formal step-by-step procedure illustrating/filming this technique has not been previously published. This protocol enables high engraftment from a low number of transplanted cells and a mechanism to sample the BM serially. Furthermore, it is possible to utilize this method to analyze the effects of injecting drugs directly into the BM cavity on the treatment of blood diseases. The procedure described here helps obtain access to BM where hematopoietic cells reside without sacrificing the mice.
This protocol is similar to the technique used for BM aspirations9. The key difference is that this paper and the accompanying video protocol detail a safe procedure for injecting cells into the marrow, whereas previous papers transplanted cells via the vein and then performed serial BM aspiration. This protocol enables successful engraftment with small numbers of a cell line (Figure 1), normal cord blood (CB)-derived HSPCs (CD34+) (Figure 2) and HSCs (CD34+CD38-CD45RA-CD90+) (Figure 3), normal BM-derived HSPCs (CD34+CD38-) (Figure 4), patient-derived leukemic stem cells (CD34+CD38-) (Figure 5), CRISPR/Cas9 gene-edited normal CB-derived HSPCs (CD34+) (Figure 6), and acute myeloid leukemia (AML)-iPSCs (Figure 7). Especially for the normal cord blood-derived HSCs, we could successfully make engraftment with only 10 cells. This method is especially valuable for experiments performed with rare or difficult-to-generate cell populations such as unmodified or gene-edited primary human acute myeloid leukemia or CB cells. Furthermore, this procedure describes an efficient method for the serial analysis of engrafted cells, which can be immediately used in downstream experiments. To avoid wasting valuable samples and ensure IF injection, we have also briefly described Akaluc-based procedure10 practices here to help solidify this technique.

<table><tbody><tr><td>1/2 mL Syringe, 27 G</td><td>BD</td><td>305620</td><td>https://www.bd.com/en-ca/offerings/capabilities/bd-luer-lok-syringe-with-attached-needle/305620</td></tr><tr><td>ACK Lysing Buffer</td><td>Qualoty Biological</td><td>118-156-101CS</td><td>https://www.qualitybiological.com/product/ack-lysing-buffer/</td></tr><tr><td>Biological Irradiator</td><td>Kimtron</td><td>IC-250</td><td>https://www.kimtron.com/ic-250</td></tr><tr><td>Chlorhexidine</td><td>Nolvasan</td><td>NDC 54771-8701-1</td><td>https://www.zoetisus.com/products/petcare/nolvasan-skin-and-wound-cleanser</td></tr><tr><td>Ethyl alcohol, proof 190</td><td>Gold Shield</td><td></td><td>https://goldsd.com/line-card/</td></tr><tr><td>FACSCanto II&amp;nbsp;</td><td>(Becton Dickinson and Company (BD), Franklin Lakes, NJ, USA</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Omega Scientific</td><td>FB-01</td><td>https://www.omegascientific.com/product/fetal-bovine-serum-usda-certified/</td></tr><tr><td>Flow cytometer, AriaII</td><td>Beckton Dickinson (BD)</td><td></td><td>cell sorting</td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440053</td><td>https://www.thermofisher.com/order/catalog/product/12440053</td></tr><tr><td>Isoflurane, USP</td><td>Dechra</td><td>NDC 17033-094-25</td><td>https://www.dechra-us.com/our-products/us/companion-animal/dog/prescription/isoflurane-usp-inhalation-anesthetic</td></tr><tr><td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice&amp;nbsp;</td><td>Jackson Laboratory, Bar Harbor, ME, USA</td><td>&lt;br/&gt; Strain #:005557</td><td>Six- to ten-week-old male or female&amp;nbsp;</td></tr><tr><td>Ophthalmic ointment, USP</td><td>Bausch Lomb</td><td>NDC 24208-780-55</td><td>https://www.bausch.com/contentassets/2914df881e4344a&lt;br/&gt; 7a202cc5a0673c977/neomycin-and-polymyxin-b-sulfates-gramicidin-ophthalmic-solution.pdf</td></tr><tr><td>OstiFen Injection (Caprofen)</td><td>VetOne</td><td>NDC 13985-748-20</td><td>http://vetone.net/Default/CatHeaderPage?id=9534ccca-eac5-4d68-8ad8-46436067587b</td></tr><tr><td>PBS, pH 7.4</td><td>Homemade</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>https://www.thermofisher.com/order/catalog/product/15140122</td></tr><tr><td>Povidone-iodine</td><td>Betadine</td><td>NDC 67618-155-16</td><td>https://www.betadine.com/veterinary-surgical-scrub-and-solution/</td></tr><tr><td>TokeOni (in the U.S.)</td><td>Sigma-Aldrich</td><td>808350-5MG</td><td>AkaLumine-HCl/Akaluc; https://www.sigmaaldrich.com/US/en/product/aldrich/808350</td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td>https://www.thermofisher.com/order/catalog/product/15575020</td></tr><tr><td>&lt;strong&gt;Engraftment antibody panel (in vivo, mouse bone marrow)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Dilution&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;: Anti-human CD45&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: V450&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: HI30</td><td>BD Biosciences</td><td>560367</td><td>1:25</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;: Anti-mouse CD45.1&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: PE-Cy7&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: A20</td><td>eBioscience</td><td>25-0453-81</td><td>1:50</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;:Anti-mouse TER-119&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: PE-Cy5&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: TER-119</td><td>eBioscience</td><td>15-5921-83</td><td>1:100</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;:Anti-human CD3&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: APC-Cy7&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: SK7</td><td>BD Biosciences</td><td>341090</td><td>1:12.5</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;:Anti-human CD19&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: APC&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: HIB19</td><td>BD Biosciences</td><td>555415</td><td>1:25</td></tr><tr><td>&lt;strong&gt;Antigen&lt;/strong&gt;:Anti-human CD33&lt;br/&gt; &lt;strong&gt;Fluorophore&lt;/strong&gt;: PE&lt;br/&gt; &lt;strong&gt;Clone&lt;/strong&gt;: WM53</td><td>BD Biosciences</td><td>555450</td><td>1:25</td></tr><tr><td>&lt;strong&gt;Live/Dead staining&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Conc.&lt;/strong&gt;</td></tr><tr><td>PI</td><td>Invitrogen</td><td></td><td>1 &amp;mu;g/mL</td></tr><tr><td>&lt;strong&gt;Compensation beads&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Negative control</td><td>BD Biosciences</td><td></td><td>See instruction for details</td></tr><tr><td>Anti-mouse Ig, &amp;kappa;</td><td>BD Biosciences</td><td></td><td>See instruction for details</td></tr></tbody></table>

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.

Following these protocols1,14,15,16,17,18,19,20, each sample was transplanted, and the xenograft mouse model was established. The aim of the experiments here was to show IF injection with several types of human cells and following bone marrow aspiration/analysis in live mice. This paper shows the results of transplantation of the following commonly used human-derived samples. Samples included the K562 cell line (Figure 1), cord blood-derived HSPCs (CD34+, Figure 2), cord blood-derived HSCs (CD34+CD38-CD90+CD45RA-, Figure 3), adult bone marrow-derived HSPCs (CD34+/CD38-, Figure 4), leukemia stem cells (CD34+/CD38-, Figure 5), CRISPR/Cas9-edited cord blood-derived HSPCs (CD34+, Figure 6), and patient-derived AML-iPSCs (Figure 7). It is important to remember that these samples are of human origin, and the variation among samples is so great that there will also be a great deal of variation in the engraftment in each mouse. To avoid wasting valuable samples when conducting large-scale experiments, it is always important to first ascertain how many cells need to be transplanted to achieve a minimum level of engraftment.
Figure 1 shows a bioluminescence image of mice intrafemorally injected with cell lines (total of 10 cells) expressing AkaLumine-HCl/Akaluc. The Akaluc signal was shown on the side of the transplanted femur. With Akaluc here and luciferase elsewhere, transplantation techniques can be easily evaluated with minimal mouse invasion. If the operator needs more confidence in their technique, we recommend practicing IF using these methods first. For more details on imaging, refer to the respective papers10 and instruction manuals. We transplanted 10 cells per mouse; three mice were transplanted, and one was successfully engrafted, represented in this figure.
Figure 2 shows a sublethally irradiated NSG mouse transplanted with 15,000 cord blood (CB)-derived normal CD34+ HSPCs (magnetic bead-enriched) and serially analyzed by IF aspiration. Although there are variations among CB donors, transplantation of 15,000 - 20,000 CD34+-enriched cells generally results in more than 10 - 20% of human CD45+ cell engraftment at 8 weeks post-transplantation (n=4). Here, a significant number of human cells were found to be engrafted at 8 weeks; most of these were considered progenitor cells and were expected to disappear gradually. A decrease in the percentage of human cells was observed at 14 and 24 weeks after transplantation. Generally, CD19+ cells predominate over CD33+ cells in NSG mice. This is the opposite of human complete blood count, where CD33+ cells predominate.
Figure 3 shows a sublethally irradiated NSG mouse transplanted with 10 cord blood-derived normal HSCs (CD34+CD38-CD90+CD45RA-) and serially analyzed by IF aspiration at 8, 12, and 16 weeks post-transplantation. Although there are variations among cord blood donors, transplantation of 5 - 10 normal HSCs generally results in 0 - 10 % human engraftment at 8 weeks post-transplantation. The proportion of CD19+ and CD33+ cells was the same as that detected with CB-derived normal HSPCs. In this situation, 50 mice were transplanted, and 18 had successfully engrafted (32.7%).
Figure 4 shows a sublethally irradiated NSG mouse transplanted with 1,000 normal adult BM-derived CD34+CD38- HSPCs. Generally, adult BM-derived HSPCs are more challenging to engraft in NSG mice compared to the same number of cord blood-derived HSPCs. Since we transplanted a low cell number, we waited for 18 weeks after the transplantation to analyze engraftment. The proportion of CD19+ and CD33+ cells was the same as that detected with CB-derived normal HSPCs (n=1).
Figure 5 shows a sublethally irradiated NSG mouse transplanted with 330 patient-derived CD34+CD38- leukemia stem cells (LSCs) following chimerism analysis by IF aspiration at 8 and 12 weeks post-transplantation. Because of the large sample-to-sample variation in primary cells, there is often a large difference in engraftment even when the same number of cells are transplanted. Therefore, some degree of optimization is significant when conducting experiments with primary AML samples. In our experience, we have transplanted 113 patient samples (bulk CD34+ or LSCs, 10-2,592 cells/mouse), and eventually made 77 samples engrafted (68.1%).
Figure 6 shows a sublethally irradiated NSG mouse transplanted with 12,000 CRISPR/Cas9 edited cells (CB-derived CD34+ -enriched) following chimerism analysis by IF aspiration at 8 and 12 weeks post-transplantation. Cells were simply edited by nucleofection of an RNP (Cas9 and sgRNA) against the targeted gene, as per the previous literature13. Here, we targeted the AAVS1 gene. The AAVS1 locus is cited as a permissive safe harbor for stable transgene expression. The proportion of CD19+ and CD33+ cells was the same level as detected in CB-derived normal HSPCs. In our experience, we have transplanted three cord blood samples (CRISPR/Cas9-edited CD34+, 12,000-62,500 cells/mouse), and eventually made 26/26 mice engrafted (100.0%).
Figure 7 shows engrafted human CD45+ cells in the BM of a secondary recipient NSG mouse. The mice were transplanted with 10,000 CD34+ cells taken from the bone marrow of human primary NSG mice transplanted with cells differentiated from AML-iPSCs17,18, and at 5 weeks, the bone marrow was examined. Despite the sample-to-sample variation, the AML-iPSCs showed stable engraftment after a second transplant (n = 7, human CD45 &#8805; 70%). Because it is a disease model, the health of the mice deteriorates as the disease progresses, but the status of the bone marrow can still be confirmed by IF aspiration at least 1-2 times.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65874/65874fig01.jpg" /> 
Figure 1: Engraftment of human leukemia cell line K562 with Akaluc. A bioluminescence image of mice intrafemorally injected with 30 K562 cells expressing Akaluc. Substrate was administrated intraperitoneally. We used a CCD camera to capture the images (10 s exposure time). The AkaLumine-HCl/ Akaluc signal was shown on the side of the transplanted femur with high signal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65874/65874fig02.jpg" /> 
Figure 2: Engraftment of long-term normal human cord blood (CB)-derived HSPCs (CD34+). Representative flow plots of engrafted human CD45+ cells from mouse intrafemorally transplanted with normal CB-derived CD34+ HSPCs (total 15,000) into sublethally irradiated NSG mouse. The human cell engraftment in the mouse was serially analyzed by BM aspirations after 8, 14, and 24 weeks of transplantation; X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. The far-right figure shows flow plots of engrafted human CD19+ and CD33+ human cells within engrafted CD45+ after 24 weeks of transplantation; X-axis: Human CD33 expression. Y-axis: Human CD19 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65874/65874fig03.jpg" /> 
Figure 3: Engraftment of long-term normal human CB-derived HSCs (CD34+CD38-CD90+CD45RA-). Representative flow plots of engrafted human CD45+ cells from mouse intrafemorally transplanted with normal CB-derived CD34+CD38-CD90+CD45RA- HSCs (total 10) into sublethally irradiated NSG mouse. The human cell engraftment in the mouse was serially analyzed by BM aspirations after 8, 12, and 16 weeks of transplantation. X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. The far-right figure shows flow plots of engrafted human CD19+ and CD33+ human cells within engrafted CD45+ after 16 weeks of transplantation; X-axis: Human CD33 expression. Y-axis: Human CD19 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65874/65874fig04.jpg" /> 
Figure 4: Engraftment of normal adult human bone marrow (BM)-derived HSPCs (CD34+CD38-) Representative flow plots of engrafted human CD45+ cells from mouse intrafemorally transplanted with normal human BM-derived CD34+CD38- cells (total 1,000) into sublethally irradiated NSG mouse. The human cell engraftment in the mouse was analyzed by BM aspirations after 18 weeks of transplantation. X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. The right figure shows flow plots of engrafted human CD19+ and CD33+ human cells within engrafted CD45+ after 18 weeks of transplantation; X-axis: Human CD33 expression. Y-axis: Human CD19 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65874/65874fig05.jpg" /> 
Figure 5: Engraftment of patient-derived leukemia stem cells (CD34+CD38-). Representative flow plots of engrafted human CD45+ cells from mouse intrafemorally transplanted with CD34+CD38- leukemic stem cells (total 330) into sublethally irradiated NSG mouse. The human cell engraftment in the mouse was serially analyzed by BM aspirations after 8 and 12 weeks of transplantation. X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65874/65874fig06.jpg" /> 
Figure 6: Engraftment of CRISPR/Cas9 edited HSPCs (CD34+). Representative flow plots of engrafted human CD45+ cells from mouse intrafemorally transplanted with normal CB-derived CD34+ -enriched CRISPR/Cas9 edited cells (total 12,000) into sublethally irradiated NSG mouse. The human cell engraftment in the mouse was serially analyzed by BM aspirations after 8 and 12 weeks of transplantation. X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. The far-right figure shows flow plots of engrafted human CD19+ and CD33+ human cells within engrafted CD45+ after 12 weeks of transplantation; X-axis: Human CD33 expression. Y-axis: Human CD19 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65874/65874fig07.jpg" /> 
Figure 7: Engraftment of patient-derived iPSCs. Representative flow plots of engrafted human CD45+ cells in the BM of a secondary recipient NSG mouse. The mice were transplanted with CD34+ cells (total 10,000) taken from the BM of human primary NSG mice transplanted with AML-iPSCs. The human cell engraftment in the mouse was analyzed by BM aspirations after 5 weeks of transplantation. X-axis: Human CD45 expression. Y-axis: Mouse CD45.1 expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/65874fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Tips for gripping the femur and finding the optimal puncture site. First, fix the femur by holding the arrowhead in the figure with the thumb and index fingers. After that, lightly prick the area circled in red in the figure several times with the needle tip to locate the center of the bone end. While feeling with the fingertips that the direction of the bone is parallel to the angle of entry of the needle, the needle is rotated like a drill as it begins to enter the bone. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/20231018_JOVE_Sup.Figures 1-5_YN 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Tips on body position when performing a mouse&#39;s bone marrow puncture (right leg). If the operatory is right-handed, the mouse is to be turned on its back, then slightly rotated to the left side to make the puncture easier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/20231018_JOVE_Sup.Figures 1-5_YN 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: Tips for finding the puncture site quickly. Once the mouse is positioned, the next step is to gently pull on the toe of the mouse to locate the white patellar ligament. Some types of mice may be difficult to recognize, so it is recommended to disinfect the skin with ethanol to make the ligament easier to see. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/20231018_JOVE_Sup.Figures 1-5_YN 3.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S4: Tips for proper gripping of the femur. Hold the area indicated by the arrowhead in the figure firmly with the thumb and forefinger, pinching the femur. The place to hold it should be a little below the ligament identified in Supplementary Figure S3, feeling the direction of the femur with the fingertips. In fatty mice, it is difficult to grasp the femur, and the fingertips move easily, so it is crucial to hold the femur so that the entire fingertip firmly wraps around the femur. While holding the toes with the right hand, avoid the skin around the knee with the left hand as much as possible so that the ligament can be easily seen. The success rate of intramedullary puncture will vary greatly depending on whether this preparation is done properly. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/20231018_JOVE_Sup.Figures 1-5_YN 4.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S5: Tips on how to find the optimal puncture site and how to insert the needle. Once the femur is firmly secured with the left index finger and thumb, as shown in Supplementary Figure S1 and Supplementary Figure S4, locate the center of the femoral epiphysis by poking the needle tip over the ligament several times.Once the puncture site is determined, rotate the needle like a drill and slowly advance the needle tip into the bone, feeling that the femur is parallel with the needle to be punctured, which is felt with the fingertips. When the needle tip is firmly in the bone marrow, resistance to the syringe suddenly disappears, which confirms that the needle has entered the bone marrow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65874/20231018_JOVE_Sup.Figures 1-5_YN 5.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis at JoVE.com

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

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. ...

simplified-intrafemoral-injections-using-live-mice-allow-for

Nanyang Technological University

Research

JoVE Journal

Medicine

1.7K Views.  Stanford University School of Medicine. Deze techniek is bedoeld om ons te helpen de biologie van normale hematopoëse en ziekte in het beenmerg beter te begrijpen door waardevolle patiëntmonsters veilig intraoraal in de muizen te injecteren en te transplanteren, gevolgd door aspiratie van het beenmerg. Het is al lang bekend dat het efficiënter is om kleine hoeveelheden waardevolle monsters rechtstreeks in het beenmerg van muizen te transplanteren voor experimenten die worden uitgevoerd door midd...

Video: Vereenvoudigde intrafemorale injecties met levende muizen maken continue beenmerganalyse mogelijk

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Cancer Research

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Developmental Biology

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 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Immunology and Infection

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

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