Name: intrafemoral injection of human hematopoietic stem and progenitor cells into immunocompromised mice
Uploaded: 2023-12-08T15:00:00.000+00:00
Duration: 3 min 40 s
Description: Read this Scientific Article Protocol about Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis at JoVE.com

The authors acknowledge the group of Dr John Dick for previous work on this method, and Monica Doedens for training. We are grateful to the Cambridge Blood and Stem Cell Biobank (CBSB), specifically Dr. Joanna Baxter and the team of CBSB nurses who consented and collected cord blood samples; our sample donors; the University Biomedical Service, specifically Nicolas Lumley and staff at The Anne McLaren Building for maintenance of our mice strains and support of our in vivo experiments; Shaaezmeen Basheer for editing of the manuscript.
E.L. is funded by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (107630/Z/15/A). L.M. is supported by Sofinter - HR Welfare Program. This research was funded in whole, or in part, by the Wellcome Trust (203151/Z/16/Z, 203151/A/16/Z, 215116/Z/18/Z) and the UKRI Medical Research Council (MC_PC_17230). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

All animal research presented here adheres to the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and was performed after ethical review and approval by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB). Female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, aged between 12 and 16 weeks (~21-30 g), bred in-house and maintained in a Specific-Pathogen-Free animal facility, were used for intrafemoral injections. De-identified CB samples were collected from healthy donors after informed consent by the Cambridge Blood and Stem Cell Biobank (CBSB) in accordance with regulated procedures approved by the Cambridgeshire Local Research Ethics Committee (18/EE/0199).&#160;
1. Preparation of the mouse
<ol>
	<li>Before irradiation, notch the ears of the mice for identification and weigh them for baseline weight.</li>
	<li>Twenty-four hours before transplantation of the cells, sub-lethally irradiate the mice with 2.4 Gy radiation.</li>
</ol>
2. Preparation of cells
NOTE: For these injections, cells can be used from fresh or frozen samples. Specific subpopulations of cells can be sorted by flow cytometry. Alternatively, cells can be cultured in the desired conditions before transplantation. For the experiments shown, we are using frozen CB CD34+ cells.
<ol>
	<li>Thaw the cells in a 50 mL tube by the dropwise addition of 10x the cell&#39;s volume of prewarmed IMDM + 50% fetal bovine serum (FBS) + 0.1 mg/mL DNase while agitating the tube manually. Centrifuge the cells at 500 &#215; g for 5 min.</li>
	<li>Resuspend the cells in 20-40 &#181;L (ideally 25 &#181;L) of PBS + Penicillin-Streptomycin (10 U/mL or 0.1%) per mouse. If possible, allow extra cells for a dead volume when taking up the cells into the syringe for injections. Store the cells on ice until injections. The maximum number of cells per injection is 4 million cells.</li>
</ol>
3. Preparation for intrafemoral injection in the animal facility
<ol>
	<li>Prepare the following three needles required for this procedure:
	<ol>
		<li>Prepare a 3 mL syringe with a 27 G 1/2&#34; needle.</li>
		<li>Prepare a 0.5 mL Insulin Syringe with 29 G x 12.7 mm needle containing the cell suspension.</li>
		<li>Prepare a 1 mL Insulin Syringe with 29 G x 0.5&#34; needle with 0.1 mg/kg buprenorphine (100 &#181;L).</li>
	</ol>
	</li>
	<li>Anesthetize the mice in an anesthetic box (through the inhalation of isoflurane 2% (v/v) and oxygen 2 L/min). Transfer the mouse to a nose cone and confirm its readiness for the procedure by toe pinch. Apply ophthalmic ointment to the eyes to prevent dryness. 
	NOTE: This procedure is carried out in a containment level 2 biosafety hood and sterile conditions are used.</li>
</ol>
4. Intrafemoral injection
<ol>
	<li>Lay the mouse on its back and have its hind leg flexed. Secure the hind leg using the non-dominant hand by placing the thumb on the foot, the middle finger on the hip, and the index finger on the outside of the femur.</li>
	<li>Gently shave or pluck the hair from around the kneecap and use an alcohol swab to wipe down the area.</li>
	<li>Use the 3 mL syringe with a 27 G 1/2&#34; needle and aim for the top inner corner of the kneecap to gently drill a hole through the skin towards the femur. Although the needle may be rotated back and forth at first, only use a clockwise motion once into the bone, until the whole needle is in the bone. 
	NOTE: The goal of this step is to generate a conduit for cell delivery. (Optional) To check if the needle is correctly in the bone, release the leg and rotate the syringe side to side; if it is correctly in the bone, the whole leg should move with the rotation. Resecure the leg using the non-dominant hand by placing the thumb on the foot, the middle finger on the hip, and the index finger on the outside of the femur, before continuing. If incorrectly inserted, the needle will likely be in the muscle or tendon, the leg will not rotate with the syringe, and you may feel the needle under the skin.</li>
	<li>Remove the needle using an anticlockwise rotation until it is halfway out. At this point, use an alcohol swab to wipe around the needle (there may be a small drop of blood) and then continue to turn the needle anticlockwise and remove the needle.</li>
	<li>Insert the 0.5 mL insulin syringe containing the cells in the femoral shaft via the same conduit. Once the needle is in, make a note of feeling a scratch indicating the correct location. At this point, release the grip on the leg, but be careful not to remove the needle from the leg. Then, gently, inject the 25 &#181;L of cell suspension into the femur and remove the needle. 
	&#8203;NOTE: The &#39;scratch&#39; described is like hitting a rough surface and if the needle feels it has gone in smoothly with no rough feeling, it is not in the correct place. Most users understand this clearly once they are successful in their practice.</li>
</ol>
5. Post injection care
<ol>
	<li>Inject the mouse subcutaneously with Buprenorphine (0.1 mg/kg, 100 &#181;L) before returning it to its cage and monitoring for recovery from the anesthetic. 
	NOTE: The mouse is not to be left unattended until it has regained sufficient consciousness to maintain sternal recumbency and is not returned to the company of other animals until fully recovered. Mice are unlikely to show any adverse effects to the intrafemoral injection; however, intrafemoral injection may result in reduced activity and pain in the operated area.</li>
	<li>Assess swelling of the hock, pain, and mobility following the procedure. 
	NOTE: Mice should regain normal limb mobility within 24 h of injection.</li>
</ol>
6. Analyzing the data
<ol>
	<li>Euthanize the mice by cervical dislocation or any approved method post injection at any time e.g., 24-48 h for homing experiments, 4-12 weeks for short-term HSC activity, &#62;18 weeks for long-term engraftment. If analysis of peripheral blood (PB) is planned, bleed the mice before sacrifice by any authorized method, yielding ideally 50-100 &#956;L of blood (no more than 10% of total volume). Remove rear leg bones and spleen according to standard dissection protocols.</li>
	<li>For bone marrow:
	<ol>
		<li>Keep the injected femur (IF) and the non-injected bones (rear leg tibias and non-injected femurs, termed BM) separate. Flush the bones using 1 mL of IMDM + 5% FBS and a 36 G x &#190;&#34; needle and centrifuge for 5 min at 500 &#215; g.</li>
		<li>Resuspend the pellet in 500 &#956;L of PBS + 3% FBS and then transfer 50 &#956;L to a well of a 96 round-bottom plate for staining.</li>
		<li>Freeze any remaining bone marrow and store it in liquid nitrogen for further ex vivo or secondary in vivo experiments.</li>
	</ol>
	</li>
	<li>For the PB:
	<ol>
		<li>Wash out the collection tube with 100 &#956;L of PBS + 3% FBS, transfer the blood to a 5 mL FACS tube, and make up the volume to 2.5 mL with PBS + 3% FBS.</li>
		<li>Carefully pipette 1 mL of Pancoll into the bottom of the FACS tubes and centrifuge for 25 min at 500 &#215; g with the brake off.</li>
		<li>Collect the buffy coat layer, transfer it to 1.5 mL tubes, and top up to 1.5 mL with PBS + 3% FBS. Centrifuge for 5 min at 500 &#215; g.</li>
		<li>Resuspend the pellet in 50 &#956;L of PBS + 3% FBS and transfer to a well of a 96 round-bottom plate for staining.</li>
	</ol>
	</li>
	<li>For the spleen:
	<ol>
		<li>Place the spleen in a cell strainer placed on a 50 mL tube and crush with the plunger from a 3 mL syringe.</li>
		<li>One milliliter at a time, add 5 mL of IMDM + 5% FBS to wash the cells through. Take 50 &#956;L to a well of a 96 round-bottom plate for staining.</li>
	</ol>
	</li>
	<li>Prepare an antibody mastermix; here is an example for 10 samples:
	<ol>
		<li>Aliquot 550 &#956;L of PBS + 3% FBS (50 &#956;L/sample + 10% extra) in a 1.5 or 2 mL tube.</li>
		<li>Add individual antibodies so that their final concentration is 2x based on their titration (e.g., for an antibody titrated 1:100, add 11 &#956;L). 
		NOTE: Choose an antibody panel that assesses potential differentiation across all blood lineages of interest, for example, CD19/FITC, GlyA/PE, CD45/PECy5, CD14/PECy7, CD33/APC, CD19/Alexa 700, CD45/BV510, and CD3/APCCy7.</li>
	</ol>
	</li>
	<li>Staining: add 50 &#956;L of antibody mastermix to each of the wells of the 96 round-bottom plate containing the samples to stain. Stain for 20 min at room temperature, add 100 &#956;L of PBS + 3% FBS to wash the cells, and centrifuge for 5 min at 500 &#215; g. Remove the supernatant.</li>
	<li>Resuspend the pellet in 200 &#956;L of PBS + 3% FBS and pass each sample through a FACS tube with a cell strainer cap. To bone marrow and spleen samples, add a further 200 &#956;L of PBS + 3% FBS.</li>
	<li>Flow cytometry controls: Make single-stain controls using compensation beads by adding 100 &#956;L of PBS, 1 drop of positive beads, 1 drop of negative beads, and 1 &#956;L of the antibody to a FACS tube. Leave for 5 min at room temperature and then add 300 &#956;L of PBS. Take 50 &#956;L of unstained bone marrow cells made up to 400 &#956;L with&#160;PBS + 3% FBS as an unstained control.</li>
	<li>Analyze the samples by flow cytometry.
	<ol>
		<li>Run single-stain controls and unstained for compensation as advised for the cytometer used. Set up gating as in Figure 3A.</li>
		<li>For PB, run all cells and for bone marrow and spleen, run a minimum of 50,000 events per sample depending on human engraftment levels.</li>
	</ol>
	</li>
</ol>

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

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	<li>Dancey, J. T., Deubelbeiss, K. A., Harker, L. A., Finch, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+kinetics+in+man.">Neutrophil kinetics in man.</a> J Clin Invest. 58 (3), 705-715 (1976).</li><li>Purton, L. E., Scadden, D. 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J., Peri&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis+in+numbers.">Hematopoiesis in numbers.</a> Trends Immunol. 42 (12), 1100-1112 (2021).</li><li>Mazurier, F., Doedens, M., Gan, O. I., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells.">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nat Med. 9 (7), 959-963 (2003).</li><li>McKenzie, J. L., Gan, O. I., Doedens, M., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+short-term+repopulating+stem+cells+are+efficiently+detected+following+intrafemoral+transplantation+into+NOD/SCID+recipients+depleted+of+CD122++cells.">Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells.</a> Blood. 106 (4), 1259-1261 (2005).</li><li>Notta, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+single+human+hematopoietic+stem+cells+capable+of+long-term+multilineage+engraftment.">Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.</a> Science. 333 (6039), 218-221 (2011).</li><li>Belluschi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelo-lymphoid+lineage+restriction+occurs+in+the+human+haematopoietic+stem+cell+compartment+before+lymphoid-primed+multipotent+progenitors.">Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.</a> Nat Commun. 9 (1), 4100 (2018).</li><li>McDermott, S. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonirradiated+NOD,B6.SCID+Il2r&#947;&#8722;/&#8722;+KitW41/W41+(NBSGW)+Mice+support+multilineage+engraftment+of+human+hematopoietic+cells.">Nonirradiated NOD,B6.SCID Il2r&#947;&#8722;/&#8722; KitW41/W41 (NBSGW) Mice support multilineage engraftment of human hematopoietic cells.</a> Stem Cell Reports. 4 (2), 171-180 (2015).</li><li>Notta, F., Doulatov, S., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+human+hematopoietic+stem+cells+is+more+efficient+in+female+NOD/SCID/IL-2Rgc-null+recipients.">Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients.</a> Blood. 115 (18), 3704-3707 (2010).</li><li>Futrega, K., Lott, W. B., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bone+marrow+HSC+transplantation+enhances+local+engraftment+at+the+expense+of+systemic+engraftment+in+NSG+mice.">Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice.</a> Sci Rep. 6, 23886 (2016).</li><li>Mende, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+molecular+and+functional+features+of+extramedullary+hematopoietic+stem+and+progenitor+cell+reservoirs+in+humans.">Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.</a> Blood. 139 (23), 3387-3401 (2022).</li><li>Medyouf, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelodysplastic+cells+in+patients+reprogram+mesenchymal+stromal+cells+to+establish+a+transplantable+stem+cell+niche+disease+unit.">Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.</a> Cell Stem Cell. 14 (6), 824-837 (2014).</li><li>Eppert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+gene+expression+programs+influence+clinical+outcome+in+human+leukemia.">Stem cell gene expression programs influence clinical outcome in human leukemia.</a> Nat Med. 17 (9), 1086-1093 (2011).</li><li>Sanchez, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+xenotransplantation+model+for+acute+myeloid+leukemia.">A robust xenotransplantation model for acute myeloid leukemia.</a> Leukemia. 23 (11), 2109-2117 (2009).</li><li>Li, J., Chen, H., Zhao, S., Wen, D., Bi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+intrafemoral+orthotopic+xenografts+of+peripheral+blood+or+bone+marrow+from+acute+myeloid+and+acute+lymphoblastic+leukemia+patients:+clinical+characterization,+methodology,+and+validation.">Patient-derived intrafemoral orthotopic xenografts of peripheral blood or bone marrow from acute myeloid and acute lymphoblastic leukemia patients: clinical characterization, methodology, and validation.</a> Clin Exp Med. 23 (4), 1293-1306 (2023).</li><li>Dobson, S. 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The authors declare no conflict of interest.

Intrafemoral injections are a useful tool in xenotransplantation when only a small number of HSPCs are available, providing improved engraftment compared to intravenous injections. However, the technique requires dexterity and training. When practicing, we would recommend using fresh cadavers of the correct weight range (see below) and injecting a colored dye (such as trypan blue) so that upon dissection, it is clear if the injection went into the femur and was restricted to it (no dye should be observed in the muscles). For this procedure, there are two critical steps: positioning and feeling the correct insertion of the second needle in the conduit. For positioning, although a method is recommended here, it is up to the individual to optimize how the mouse leg is most comfortably and reproducibly held. For example, a member of our team prefers to secure the leg from across the body. The key in any circumstance is that the leg, and therefore, femur, is secure. The &#39;scratch&#39; described to confirm whether the second needle is correctly inserted in the femur, is like hitting a rough surface but is difficult to explain further in writing. It is usually felt very clearly by most users once they are successful in their practice.
In the context of transplantation of human HSCs, and following a comparison of four immunocompromised mice, NSG mice are most commonly used for this technique9. Nonetheless, since the publication of that article in 2010, other immunocompromised mice have been generated that can be utilized with intrafemoral injections such as NOD.CgPrkdcscidIl2rgtm1WjlTg(CMVIL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3)10 and CSF1h/h IL3/CSF2h/hSIRPAtg THPOh/h Rag2&#8722;/&#8722; Il2rg&#8722;/ (MISTRG)11, both producing human cytokines to support human hematopoiesis in mice. Alternatively, the need for irradiation may be overcome by the use of NOD,B6.SCID Il2r&#947;&#8722;/&#8722;KitW41/W41 (NBSGW) mice12.
This method does come with its limitations and considerations. As mentioned in the introduction, this method is not useful if the microenvironment of the bone is of interest to the research as it is damaged in the process of irradiation and injection. Additionally, the lineage output of the cells can vary depending on the mouse model. For NSG mice, an improvement of erythroid engraftment can be achieved if the mice are treated with human erythropoietin8. The age and sex of the mice must also be considered. If the mice are too young and therefore small, there is a risk of breaking the bone during the procedure and if the mice are older and bigger, it can be hard to drill the initial hole. We recommend mice between 21 g and 30 g in weight. Furthermore, as it has been published that for NSG mice, engraftment is improved in female mice compared to males13, we preferentially use females over males. Due to the potentially higher engraftment observed after intrafemoral injections compared to intravenous injections, the number of mice needed to obtain detectable measurements and statistically significant results may be lower. Caution should be taken with cell doses used, as high doses (&#62;5 &#215; 104 CD34+ cells from CB) may lead to saturation of the injected bone at the expense of systemic engraftment14.
To date, our laboratory has used intrafemoral injections for the xenotransplantation of healthy CB8, spleen, and PB15 cells. However, many other studies have shown their use can be beneficial for the transplantation of hematological samples from patients suffering from various hematological diseases. For example, Medyouf and colleagues found that the addition of patient-derived mesenchymal stromal cells improved engraftment of CD34+ cells from patients with Myelodysplastic syndromes after intrafemoral injection16. Intrafemoral injections are also commonly employed to test leukemic engraftment of Acute Myeloid Leukemia17,18 and Acute Lymphoblastic Leukemia samples19,20. Finally, intrafemoral injections have been used in a recent clinical trial of hematopoietic stem cell gene therapy of adult and pediatric patients affected by transfusion-dependent &#223;-thalassemia21, demonstrating their clinical relevance.

Blood has one of the highest regeneration rates in the human body, producing 1 &#215; 1012 cells per day in the adult human bone marrow1. Hematopoietic stem cells (HSCs) guarantee blood production over the lifespan by the process of hematopoiesis and are defined by their capacity to produce all blood cell types (multipotentiality) while maintaining themselves (self-renewal). Historically, the gold standard for testing the function of an HSC has always relied on transplantation, testing the ability of a donor population to reconstitute all blood lineages of a mouse long-term (commonly defined as a minimum of 20 weeks)2. A large body of functional work spanning several decades has demonstrated that the HSC compartment is heterogeneous in both lineage output and long-term reconstitution. The toolkit to study hematopoiesis has expanded considerably over the years, with many new techniques, including in vitro single-cell functional assays, single-cell -omics approaches, and lineage tracing3. The latter have conclusively demonstrated that the contributions of HSC and multipotent progenitors largely differ in native hematopoiesis and under the stress imposed by transplantation. All these techniques complement transplantation assays, which remain important to assess the long-term repopulation capacity of HSCs. In the context of the study of human hematopoiesis, xenotransplantation provides the only method to experimentally assess self-renewal in a whole-organism setting.
Xenotransplantation of HSCs is commonly performed using intravenous injection of cells into immunocompromised mice. However, HSCs are rare4 and access to human samples containing HSCs is limited. In 2003, the group of John Dick adapted a protocol for bone marrow aspiration and intrafemorally injected non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice with Lin&#8722;CD34+ umbilical cord blood (CB) cells5. To our knowledge, there has been no reported formal comparison of intravenous versus intrafemoral injections in long-term and serial transplantation outcomes. However, compared directly with intravenous injections, intrafemoral injections provide larger graft sizes with the same number of transplanted cells6, at least in the short term. In addition, engraftment can be detected with many fewer hematopoietic stem and progenitor cells (HSPCs) transplanted. This is thought to be because intrafemoral delivery bypasses the need for HSCs to home to the bone marrow, which in the xenograft context is limiting due to a lack of cross-species reactivity for a number of receptors and cytokines. Via the use of intrafemoral injections, Notta and colleagues were the first to transplant single human HSCs7, though extra considerations need to be taken, as described in their methods. Intrafemoral delivery of HSPCs also has limitations. The injection itself disrupts and destroys part of the bone marrow, and therefore is not indicated for studies of the crosstalk between HSCs and their bone marrow microenvironment. Additionally, the maximum number of cells is limited by the volume of that bone cavity and that may be too few for some applications. As with every technique, its application in a specific experiment needs to be weighed up based on the benefits/disadvantages and the question being asked. In the context of xenotransplantation, if the aim of the experiment is to test the engraftment of a low number of human HSPCs with no assessment of microenvironment, intrafemoral delivery is usually preferred over intravenous injection.

<table><tbody><tr><td>0.5 mL Insulin Syringe with 29 G x 12.7 mm Needle</td><td>BD</td><td>324892</td><td></td></tr><tr><td>1 mL Insulin Syringe with 29 G x 0.5&quot; Needle</td><td>BD</td><td>324827</td><td></td></tr><tr><td>1.5 mL tube</td><td>Fisherbrand</td><td>509-GRD-PFB</td><td></td></tr><tr><td>3 mL syringe&amp;nbsp;</td><td>HENKE SASS WOLF GMBH</td><td>4020.000V0</td><td></td></tr><tr><td>5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Cap 12 x 75 mm</td><td>Falcon</td><td>352235</td><td>FACS tube</td></tr><tr><td>5 mL Round Bottom Polystyrene Test Tube with Snap Cap 12 x 75 mm</td><td>Falcon</td><td>352058</td><td>FACS tube</td></tr><tr><td>27 G 1/2&quot; needle&amp;nbsp;</td><td>BD&amp;nbsp;</td><td>300635</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer for 50 mL tube</td><td>Greiner Bio-one</td><td>542040</td><td></td></tr><tr><td>50 mL tube</td><td>Sarstedt Ltd</td><td>62.547.254</td><td></td></tr><tr><td>96 well round-bottom plate&amp;nbsp;</td><td>Falcon</td><td>351177</td><td></td></tr><tr><td>Alcohol Swab</td><td>VITREX MEDICAL A/S</td><td>520213</td><td></td></tr><tr><td>BD LSR Fortessa X-20 Cell Analyzer&amp;nbsp;</td><td>BD</td><td></td><td>flow cytometer</td></tr><tr><td>Buphrenorphine</td><td>Animalcare</td><td>XVD190</td><td></td></tr><tr><td>CD14/PECy7 (Clone M5E2)</td><td>biolegend</td><td>301814</td><td>Used at 1 in 1000</td></tr><tr><td>CD19/Alexa 700 (Clone HIB19)</td><td>biolegend</td><td>302226</td><td>Used at 1 in 300</td></tr><tr><td>CD19/FITC (Clone HIB19)</td><td>biolegend</td><td>302206</td><td>Used at 1 in 200</td></tr><tr><td>CD3/APCCy7&amp;nbsp;(Clone HIT3a)</td><td>biolegend</td><td>300318</td><td>Used at 1 in 100</td></tr><tr><td>CD33/APC (Clone P67.6)</td><td>BD</td><td>345800</td><td>Used at 1 in 200</td></tr><tr><td>CD45/BV510 (Clone HI30)</td><td>biolegend</td><td>304036</td><td>Used at 1 in 500</td></tr><tr><td>CD45/PECy5 (Clone 2D1)</td><td>biolegend</td><td>368526</td><td>Used at 1 in 300</td></tr><tr><td>CompBeads Anti-Mouse Ig, &amp;kappa;/Negative Control Compensation Particles Set</td><td>BD</td><td>552843</td><td></td></tr><tr><td>Dnase 1</td><td>Worthington Biochemical</td><td>LS002139</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>PAN-Biotech</td><td>P40-37500</td><td></td></tr><tr><td>Glycophorin A (GlyA)/PE (Clone GA-R2)</td><td>BD</td><td>340947</td><td>Used at 1 in 1000</td></tr><tr><td>Iscove Modified Dulbecco Media (IMDM)</td><td>PAN-Biotech</td><td>P04-20250</td><td></td></tr><tr><td>Isoflurane (IsoFlo 100% w/w Inhalation Vapor, liquid)</td><td>Zoetis</td><td>115095</td><td></td></tr><tr><td>Microvette&amp;nbsp;300 Lithium heparin LH, 300 &amp;micro;L</td><td>Sarstedt Ltd</td><td>20.1309</td><td>Mouse blood collection tube</td></tr><tr><td>NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice&amp;nbsp;</td><td>Charles River&amp;nbsp;</td><td></td><td></td></tr><tr><td>Pancoll human, Density: 1.077 g/mL</td><td>PAN-Biotech</td><td>P04-60500</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190169</td><td></td></tr></tbody></table>

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.

The engraftment of the intrafemorally injected cells can be assessed at any time point from 24 h onwards depending on the experimental design. At the end time point, IF, BM, PB, and spleens may be collected. These can be processed, and the level of engraftment assessed via flow cytometry. To robustly call human engraftment even at low levels, we stained with two distinct antibodies against human CD45 (clone HI30 and clone 2D1). Only cells positive for both antibodies (CD45++) were considered of human origin. The threshold of engraftment was set as follows: (% CD45++ + % GlyA+) &#8805; 0.01 % and a minimum of 30 cells recorded in both gates (Figure 1)7,8. Migration of human cells to bones other than the one injected is usually delayed. Therefore, if the bones are harvested before 8 weeks, it is likely that the engraftment of the IF is higher than the BM (Figure 2A). This tends to equalize after 8 weeks (Figure 2B,C). Engraftment levels in PB are expected to be variable (Figure 2D).
All bone marrow cells were stained using an antibody panel that assesses the level of engraftment across different blood lineages: CD19/FITC, GlyA/PE, CD45/PECy5, CD14/PECy7, CD33/APC, CD19/Alexa 700, CD45/BV510, and CD3/APCCy7. To assess which blood lineages are present in the human graft, we used the following thresholds: Lymphoid (Ly) lineage: CD45++ CD19++ (positive for two different CD19 antibodies) and/or&#160;CD45++ CD3+ &#8805; 20 cells; Myeloid (My) lineage: CD45++ CD33+ &#8805; 20 cells; Erythroid (Ery) lineage: CD45&#8722; GlyA+ &#8805; 20 cells (Figure 3A). Lineage composition is usually similar between IF and BM, but in certain instances, mature myeloid cells can be slightly but significantly increased in BM compared to IF (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66315/66315fig01.jpg" /> 
Figure 1: Representative human engraftment in the injected femur, other hind leg bones, and peripheral blood of mice intrafemorally injected with human hematopoietic stem and progenitor cells. Representative flow plots of non-engrafted (Mouse 1) and engrafted (Mice 2 and 3) mice. Mice are counted as engrafted when (% CD45++ + % GlyA+) &#8805; 0.01% and a minimum of 30 cells were recorded in both gates. Mouse 2 demonstrates a case where the engraftment in the BM is lower than the IF. Mice were transplanted with CB CD34+ HSPCs and tissues were analyzed at 12 weeks. Abbreviations: IF = injected femur; BM = bone marrow (other hind leg bones). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66315/66315fig01large.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/66315/66315fig02.jpg" /> 
Figure 2: Comparison of engraftment in the injected femur and other hind leg bones at 4, 8, and 20 weeks post intrafemoral injection. (A-C) Size of the human graft (% CD45++ + % GlyA+) in IF and BM (left panels) and the ratio of engraftment in BM:IF (right panels) at 4 weeks (A, n = 5 mice), 8 weeks (B, n = 7 mice), and 20 weeks (C, n = 11 mice) post transplantation. Mice were engrafted with 100, 500, or 3,000 Subset 1 (CD19-CD34+CD38-CD45RA-CD34loCLEC9Ahi) or Subset 2 (CD19-CD34+CD38-CD45RA-CD34hiCLEC9Alo) cells. Cell type and dose depicted by different symbols (see legend). Mice shown below the black dashed line are considered non-engrafted as their graft size falls below the threshold ([% CD45++ + % GlyA+] &#8805; 0.01% and at least 30 cells recorded). CD45++ cells are positive for two distinct CD45 antibodies. Ratio of engraftment BM:IF for high dose mice only, 500 Subset 1 cells (A n = 3, B n = 4) or 3,000 Subset 1 cells (C n = 10). **p &#60; 0.01 by one-sample t-test. (D) Percentage human engraftment (% CD45++ + % GlyA+) in PB across time. Mice were engrafted with 3,000 Subset 1 (D, n = 10 mice). This is a re-analysis of the data published in Belluschi et al.8. Abbreviations: IF = injected femur; BM = bone marrow (other hind leg bones), PB = peripheral blood. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66315/66315fig02large.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/66315/66315fig03.jpg" /> 
Figure 3: Analysis of lineage potential in vivo. (A) Representative example of the gating strategy used to assess lineage potential in vivo. My lineage: CD45++ CD33+ &#8805; 20 cells; Ly lineage: CD45++ CD19++ (positive for two distinct CD19 antibodies) and/or&#160;CD45++ CD3+ &#8805; 20 cells; Ery lineage: CD45&#8722; GlyA+ &#8805; 20 cells. (B) Percentage of human Ly, My, and Ery engraftment in the injected femur and other hind leg bones 20 weeks post transplantation of mice engrafted with 3000 Subset 1 (CD19-CD34+CD38-CD45RA-CD34loCLEC9Ahi). Only mice with percentage human engraftment (% CD45++ + % GlyA+) &#62; 1% shown. *p &#60; 0.05 by paired t-test. This is a re-analysis of the data published in Belluschi et al.8. Abbreviations: My = myeloid; Ly = lymphoid; Ery = erythroid; FSC-A = forward scatter area; FSC-W = forward scatter width; PE = Phycoerythrin; APC =Allophycocyanin; IF = injected femur; BM = bone marrow (other hind leg bones). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66315/66315fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis at JoVE.com

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.

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 ...

intrafemoral-injection-human-hematopoietic-stem-progenitor-cells-into

Nanyang Technological University

Research

JoVE Journal

Biology

871 Views.  University of Cambridge. Onze interesse gaat uit naar het bestuderen van gezonde menselijke hematopoëse gedurende een heel leven, van de foetus tot ouderen. We bestuderen bijvoorbeeld de lever bij de foetus en het beenmerg en perifeer bloed bij de volwassene. We denken dat het belangrijk is om de gezonde context te bestuderen om ons te helpen begrijpen wat er misgaat bij ziekte.Er zijn veel eencellige Onyx-benaderingen die het veld de afgelopen jaren vooruit hebben geholpen, ...

Video: Auteur in de schijnwerpers: onderzoek naar de levensduurdynamiek van gezonde menselijke hematopoëse

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Cancer Research

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 ...

Medicine

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Immunology and Infection

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

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

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

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

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

Bioengineering

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) ...

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. ...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Source: Lanis, J. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

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

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