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

<ol>
	<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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+factors+in+murine+hematopoietic+stem+cell+assays.">Limiting factors in murine hematopoietic stem cell assays.</a> Cell Stem Cell. 1 (3), 263-270 (2007).</li><li>Laurenti, E., G&#246;ttgens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+haematopoietic+stem+cells+to+complex+differentiation+landscapes.">From haematopoietic stem cells to complex differentiation landscapes.</a> Nature. 553 (7689), 418-426 (2018).</li><li>Cosgrove, J., Hustin, L. S. P., de Boer, R. 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. P., Eppert, K., Lechman, E. R., 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=Comparison+of+human+cord+blood+engraftment+between+immunocompromised+mouse+strains.">Comparison of human cord blood engraftment between immunocompromised mouse strains.</a> Blood. 116 (2), 193-200 (2010).</li><li>Billerbeck, 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=Development+of+human+CD4+FoxP3++regulatory+T+cells+in+human+stem+cell+factor-,+granulocyte-macrophage+colony-stimulating+factor-,+and+interleukin-3-expressing+NOD-SCID+IL2R&#947;(null)+humanized+mice.">Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2R&#947;(null) humanized mice.</a> Blood. 117 (11), 3076-3086 (2011).</li><li>Rongvaux, A., 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=Development+and+function+of+human+innate+immune+cells+in+a+humanized+mouse+model.">Development and function of human innate immune cells in a humanized mouse model.</a> Nat Biotechnol. 32 (4), 364-372 (2014).</li><li>McIntosh, B. 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. M., 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=Relapse-fated+latent+diagnosis+subclones+in+acute+B+lineage+leukemia+are+drug+tolerant+and+possess+distinct+metabolic+programs.">Relapse-fated latent diagnosis subclones in acute B lineage leukemia are drug tolerant and possess distinct metabolic programs.</a> Cancer Discov. 10 (4), 568-587 (2020).</li><li>Marktel, 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=Intrabone+hematopoietic+stem+cell+gene+therapy+for+adult+and+pediatric+patients+affected+by+transfusion-dependent+&#223;-thalassemia.">Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent &#223;-thalassemia.</a> Nat Med. 25 (2), 234-241 (2019).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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&#160;</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&#34; needle&#160;</td>
			<td>BD&#160;</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;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&#160;</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&#160;</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&#160;(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, &#954;/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&#160;300 Lithium heparin LH, 300 &#181;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&#160;</td>
			<td>Charles River&#160;</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&#160;</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

835 Views.  University of Cambridge. 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.

Video: Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

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 ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

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) ...