We were supported with expert technical assistance by the HSCI-CRM Flow Cytometry facility at Massachusetts General Hospital and the Bauer Core Facility at Harvard University. T.K and K. G were supported by the Swedish Research Council and C.M. by the German Research Foundation. We thank Sergey Isaev and I-Hsiu Lee for assistance in analysis of single-cell RNA sequencing data.

All work with human tissue was conducted after approval by the Massachusetts General Hospital Internal Review Board (IRB). All animal procedures were conducted in accordance with the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) guidelines. C57Bl/6 mice, 8-10 weeks old, and both males and females, were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Preparation of murine thymic tissue
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>Medium 199 (M199) with 2% (v/v) fetal bovine serum (FBS) (see Table of Materials).</li>
		<li>Murine dissociation cocktail: 0.5 WU/mL Liberase TM and 6.3 U/mL DNase I in M199 + 2% FBS (see Table of Materials).</li>
		<li>M199 with 2% (w/v) Bovine serum albumin (BSA). 
		NOTE: FBS containing medium can be switched to BSA to improve the RNA quality.</li>
	</ol>
	</li>
	<li>Perform the murine thymus dissection
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation (following institutionally approved protocols) and place it on its back. Wet the fur on the chest by spraying 70% ethanol and proceed to open the chest cavity.</li>
		<li>Carefully dissect the thymus by first making a transverse incision just below the rib cage with surgical scissors. Cut the ribs on each side of the mouse all the way up to the clavicle. Then cut the diaphragm so that the chest wall can be released. Lift the ribs with a pair of forceps to expose the thoracic cavity.
		<ol>
			<li>The thymus is a white, bilobular organ, sitting at the top of the cavity, just above the heart. Hold the thymus with the forceps and gently cut the connective tissue that holds the thymus in place. Put the dissected thymic tissue in the well of a 6-well plate with M199 + 2% FBS on ice. 
			NOTE: When working with aged or radiation-conditioned mice or mutant strains with thymic defects, the thymus may be so small that a dissection microscope is required to reliably excise the organ.</li>
		</ol>
		</li>
		<li>Using a dissection microscope, remove any extrathymic tissue using blunt-ended forceps and micro-spring scissors.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine thymic tissue (30 min in increments of 10 min)
	<ol>
		<li>Place the cleaned thymus in the cap of a 15 mL tube and finely mince the tissue with sterile surgical scissors. 
		NOTE: Mincing the tissue in the cap of the tube that is to be used for the digestion minimizes tissue loss due to transfer from one container to another. This is very important when working with aged or radiation-conditioned mice or mutant strains with thymic defects. The small thymus size in these settings necessitates the need to reduce the loss of material wherever possible.</li>
		<li>Add 2 mL of murine dissociation cocktail to the 15 mL tube, attach the cap containing the thymus pieces, and invert 5 times to ensure that the tissue is resuspended in the dissociation cocktail.</li>
		<li>To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 &#176;C water bath with 250 rpm shaking. Incubate for 10 min. 
		NOTE: The success of the digestion protocol depends on the incubation being performed at 37 &#176;C and with agitation. The described protocol has been optimized using a shaking water bath. If that is not available, other approaches that enable agitation in a heated setting might work, but we recommend that the protocol be optimized for these settings first.</li>
		<li>Place the 15 mL tube in a rack and let the tissue pieces settle. Carefully remove the supernatant and pass it over a 70 &#181;m cell strainer into an ice-cold 50 ml tube.</li>
		<li>Repeat steps 2-4 twice for a total of 3 rounds of digestion. The tissue should be more or less completely dissociated by the end of the last incubation.</li>
		<li>Add 20 mL of M199 + 2% FBS to the collected cell suspension. Spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend in an appropriate volume of M199 + 2% FBS. Then proceed to cell counting. 
		NOTE: The volume of M199 + 2% FBS to resuspend in will depend on the state of the thymus. For 6-10-week-old mice, we recommend resuspending the tissue in 3-4 mL of M199 + 2% FBS. However, for experiments using aged or radiation-conditioned tissue or mutant strains with thymic defects, the small thymus size will require lower volumes ranging from 0.1-1 mL.</li>
		<li>Count cells by diluting the cell suspension 1:2 in 0.4% (w/v) trypan blue solution. Load cells onto a hemocytometer and count under a bright-field microscope. Count unstained cells to determine the number of live cells per milliliter and the number of blue-stained cells to assess sample viability.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of murine thymic stroma
	<ol>
		<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C. Resuspend cells at a concentration of 5 x 107 cells/mL in M199 + 2% FBS supplemented with 25 U/mL Protector RNase inhibitor (see Table of Materials).</li>
		<li>Add murine Fc Block (see Table of Materials) at a concentration of 2 &#181;g/mL and incubate for 10 min at 4 &#176;C.</li>
		<li>Stain the cells with the following antibodies: CD45-PE/Cy7 (4 &#181;g/mL), Ter119-PE (4 &#181;g/mL), CD31-BUV737 (2 &#181;g/mL), and EpCam-BV711 (2 &#181;g/mL) (see Table of Materials) for 30 min at 4 &#176;C.</li>
		<li>Wash the samples in M199 + 2% BSA and spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove supernatant and resuspend cells in M199 + 2% BSA containing a viability dye such as DAPI (0.5 &#181;g/mL) or 7-AAD (1 &#181;g/mL) (see Table of Materials). Proceed to FACS isolation of thymic stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>
2. Preparation of human thymic tissue
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>Medium 199 (M199) with 2% (v/v) FBS.</li>
		<li>M199 with 1.5% (w/v) BSA.</li>
		<li>Human dissociation cocktail: 1 mg/ml Collagenase IV and 2 mg/mL Dispase and 6.3 U/mL DNase I in M199 +1.5% BSA (see Table of Materials). 
		NOTE: The last digestion step in the preparation of human thymic tissue includes trypsin. It is therefore essential that all steps up until that point are carried out in BSA containing buffer as FBS will inhibit trypsin activity.</li>
	</ol>
	</li>
	<li>Perform human thymic tissue isolation
	<ol>
		<li>Place the human thymus (collected following previously published method19) in a suitable volume of M199 + 1.5% BSA and store it on ice until processing starts.</li>
		<li>While working with human thymic tissue, handle it as if it is potentially infectious by carrying out the work in a tissue culture hood and using standard precautions per CDC guidelines20.</li>
		<li>Put the tissue in a 10 cm Petri dish with 10 mL of ice-cold M199 + 1.5% BSA. Using sterile forceps and scissors, carefully clean away extrathymic fat or tissue that was damaged as the organ was extracted.</li>
		<li>Using surgical scissors and a pair of forceps, cut the thymus into 1 cm3 cubes and use the plunger of a 10 mL syringe to gently push down on the pieces. This will release some of the developing thymocytes, thus reducing the volume of tissue to be digested. 
		NOTE: Save the released hematopoietic fraction if subsequent analysis of thymocyte developmental stages is needed. However, do not combine this fraction with the stromal cell fraction generated during enzymatic digestion. It was observed that pooling the two fractions significantly increases the risk of clogs forming in the cell suspension, ultimately reducing the yield of stromal cells. Few stromal cells are lost during the gentle crushing of the tissue.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of human thymic tissue (90 min in increments of 30 min)
	<ol>
		<li>Transfer approximately 5 mg of the lightly crushed thymic tissue pieces to the cap of a 50 mL conical tube. Then cut the tissue finely using sterile surgical scissors.</li>
		<li>Pipette 8 mL of human dissociation cocktail into the 50 mL tube, attach the cap containing the minced human thymic tissue, and then gently invert the tube 5 times to ensure that all the tissue is mixed in with the enzyme cocktail.</li>
		<li>Secure the cap of the tube with paraffin film to prevent leakage. Incubate the tube horizontally in a shaking (250 rpm) water bath at 37 &#176;C for 30 min.</li>
		<li>Place the 50 mL tube in a rack and let the human thymic tissue pieces settle before removing the supernatant and passing it over a 70 &#181;m cell strainer into a new 50 ml tube placed on ice.</li>
		<li>Repeat steps 2.3.2-1.3.4 for a total of two rounds of digestion with the human dissociation cocktail.</li>
		<li>After the second round of digestion is done, add an additional 8 mL of the human dissociation cocktail to the remaining tissue. Then also pipette 2 mL of 0.25% trypsin into the tissue dissociation tube. Incubate for an additional 30 min at 37 &#176;C, shaking at 250 rpm. 
		NOTE: It is absolutely essential that the digestion time for human tissue is at least 90 min, as shorter incubation times lead to drastically reduced stromal cell yields.</li>
		<li>Once the last incubation step is done, pool the cell suspension and remaining tissue fragments with the supernatant collected in previous steps by passing over a 70 &#181;m cell strainer. Add 3 mL of FBS to the sample to break the trypsin reaction and place on ice.</li>
		<li>Proceed to counting the cells as described in step 1.3.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of human thymic stroma
	<ol>
		<li>Spin down the cell suspension of human thymus and resuspend at a concentration of 5 x 107 cells/mL in M199 + 2% FBS supplemented with 25 U/mL Protector RNase inhibitor.</li>
		<li>Incubate with human Fc Block (5 &#181;g/mL) (see Table of Materials) for 10 min at 4 &#176;C.</li>
		<li>Stain the sample for 30 min at 4 &#176;C with CD45-BV711 (2.5 &#181;g/mL), CD235a-BV711 (2.5 &#181;g/mL), Lineage-cocktail-FITC (3.76 &#181;g/mL), CD66b-FITC (20 &#181;L/100 &#181;L sample), CD8-APC/Cy7 (5 &#181;L/100 &#181;L sample), CD4-BV605 (5 &#181;L/100 &#181;L sample), CD31-PE/Dazzle594 (10 &#181;g/mL), and EpCam-BV421 (2.5 &#181;g/mL) (see Table of Materials).</li>
		<li>Wash the samples in M199 + 1.5% BSA and spin down at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend cells in M199 + 1.5% BSA containing a viability dye such as DAPI (0.5 &#181;g/mL) or 7-AAD (1 &#181;g/mL). Proceed to FACS isolation of thymic stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>
3. Preparation of murine bone and bone marrow tissue
NOTE: Bone and bone marrow fractions are prepared in two separate digestion reactions to obtain maximum purity of stromal cells and optimal dissociation of tissues. The samples can be pooled after the digestion steps to be sorted as one stromal compartment.
<ol>
	<li>Prepare the following buffers and solutions.
	<ol>
		<li>M199 with 2% (v/v) FBS.</li>
		<li>Murine B&#38;M (bone and marrow) dissociation cocktail: Stemxyme 2 mg/mL, Dispase 1 mg/mL and 10 U/mL DNase I (see Table of Materials) in M199 +2% FBS. 
		NOTE: Prepare B&#38;M dissociation mix fresh and keep mix on ice until use.</li>
		<li>M199 with 0.5% (w/v) BSA.</li>
		<li>Phosphate-buffered saline (PBS) with 0.5% (w/v) BSA.</li>
		<li>M199 with 2% (w/v) Bovine serum albumin (BSA) and 2 mM EDTA. 
		NOTE: FBS containing medium can be switched to BSA to improve RNA quality.</li>
	</ol>
	</li>
	<li>Perform murine bone marrow separation
	<ol>
		<li>Euthanize the mouse by CO2 asphyxiation (following institutionally approved protocols). Spray with 70% ethanol to wet the fur.</li>
		<li>Carefully remove the femur, tibia, pelvis, and humerus from each side and place them in a well containing M199 + 2% FBS on ice21,22.</li>
		<li>Remove all muscle, ligament, and cartilage tissue with tissue wipes to minimize damage to the bone and preserve the periosteal layer. Place them in a new well with M199 + 2% FBS on ice.</li>
		<li>Use a scalpel and cut through the growth plates of the long bones to remove the epiphysis and place it in a 10 cm dish with M199 + 2% FBS on ice.</li>
		<li>Flush the bone marrow out of the bones until pale white. Use a 28 G needle and a 10 mL syringe filled with 10 mL M199 + 2% FBS to flush the marrow into a 15 mL tube on ice. Place the flushed bone in the 10 cm dish with the epiphysis. 
		NOTE: Complete flushing of the bone marrow for entire separation of tissues will result in better sample preparation.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine bone marrow tissue (total 30 min in increments of 10 min)
	<ol>
		<li>Leave the 15 mL tube standing upright on ice until the marrow sinks to the bottom of the tube. Slowly remove the 10 mL M199 + 2% FBS by pipetting.</li>
		<li>Add 4 mL of B&#38;M dissociation cocktail to the 15 mL tube and place it in a 37 &#176;C water bath (no shaking) for 10 min.</li>
		<li>After 5 min, invert the tube three times and place it back in the water bath.</li>
		<li>After the first 10 min, ensure the pellet is at the bottom and collect the supernatant while filtering through a 70 &#181;m cell strainer into an ice-cold 50 mL tube. Add 2 mL of fresh B&#38;M dissociation cocktail to the remaining BM pellet and place it back in the water bath.</li>
		<li>Dissociate any remaining bone marrow pieces by vigorous pipetting using a 1 mL pipette. Collect all the resulting cell suspension in the same tube as in the previous steps. Add 20 mL of M199 + 0.5% BSA + 2 mM EDTA to the collected cell suspension. 
		NOTE: In step 3.3.5, the buffer added after the final digested sample contains EDTA to aid in inhibiting further enzymatic activity.</li>
	</ol>
	</li>
	<li>Perform hematopoietic cell depletion of murine bone marrow tissue.
	<ol>
		<li>Centrifuge cells at 500 x g for 5 min at 4 &#176;C. Resuspend in 500 &#181;L of PBS + 0.5% BSA with biotin antibodies (5 mg/mL) targeting CD45, Ter119, CD11b, Gr1, B220, and CD3 (see Table of Materials). Transfer to a round-bottom tube with a cap.</li>
		<li>Incubate on an orbital shaker for 10 min at room temperature in the dark.</li>
		<li>Vortex the magnetic beads thoroughly and add 25 &#181;L of magnetic beads to the tube.</li>
		<li>Incubate on an orbital shaker for 5 min at room temperature in the dark.</li>
		<li>Place the tube in the matching magnet for 2-3 min and collect the supernatant in a fresh tube. 
		NOTE: The bone marrow can be pooled or kept separate from the bone fraction after this step. If the lineage depletion is successful, the resulting cell suspension should be devoid of red cells.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of murine bone tissue.
	<ol>
		<li>Break the bones into smaller pieces in the 10 cm dish with the flat cap-end of a 50 mL tube. Filter the supernatant through a 70 &#181;m cell strainer to isolate bone fragments inside the filter. 
		NOTE: Alternatively, a mortar and pestle can be used for bone fragmentation. This option, however, tends to generate more debris and lower stromal cell viability when the bone is ground rather than broken. The stroma cells are still attached to and in the bone, and the supernatant containing hematopoietic cells is to be discarded.</li>
		<li>Use scissors to cut the bone fragments in the cell strainer. This will increase the surface area for a more efficient enzymatic dissociation. Wash the bone with 5 mL of M199 + 2% FBS.</li>
		<li>Place the bone fraction in 5 mL of murine B&#38;M dissociation (see Table of Materials) mix in a new 50 mL tube.</li>
		<li>To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 &#176;C water bath with 120 rpm shaking for 30 min.</li>
		<li>After the digestion, add 25 mL of M199 + 0.5% BSA + 2 mM EDTA and filter the supernatant containing the stromal cells through a 70 &#181;m cell strainer into an ice-cold 50 ml tube. 
		NOTE: Check that the resulting bone fragments have been visibly diminished by the digestion. In step 3.5.5, the buffer added after the final digested sample contains EDTA to aid in inhibiting further enzymatic activity.</li>
	</ol>
	</li>
	<li>Perform fluorescence activated cell sorting of murine bone and bone marrow stroma.
	<ol>
		<li>Spin down the cells at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend in murine Fc Block at a concentration of 10 &#181;g/mL in PBS + 0.5% BSA + 2 mM EDTA and incubate for 10 min at 4 &#176;C.</li>
		<li>Stain the cells (up to 10 million cells/100 &#181;L total staining volume) with the following antibodies: CD45-PE/Cy7 (1 &#181;g/mL), Ter119-PE/Cy7 (1 &#181;g/mL), CD31-BV421 (0.66 &#181;g/mL), CD140a-APC (2 &#181;g/mL), Sca1-AF700 (0.66 &#181;g/mL), CD51-PE (2 &#181;g/mL), CD105-PE/dazzle (2 &#181;g/mL) (see Table of Materials).</li>
		<li>Leave Calcein-AM to equilibrate to room temperature and resuspend in 50 &#181;L of DMSO. Prepare fresh for every use. Keep at room temperature after resuspension. Pre-dilute the calcein in PBS (0.1 mg/mL) before adding to the sample (20 &#181;g/mL) already resuspended in the antibody staining cocktail and incubate for 20-30 min at 4 &#176;C in the dark.</li>
		<li>Wash the samples in PBS + 0.5% BSA + 2 mM EDTA and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend the cells in PBS + 0.5% BSA + 2 mM EDTA. Proceed to FACS isolation of bone and bone marrow stromal cells following established protocols19.</li>
	</ol>
	</li>
</ol>

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

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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=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kode, 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=Leukaemogenesis+induced+by+an+activating+beta-catenin+mutation+in+osteoblasts.">Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts.</a> Nature. 506 (7487), 240-244 (2014).</li><li>Agarwal, P., 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=Mesenchymal+niche-specific+expression+of+cxcl12+controls+quiescence+of+treatment-resistant+leukemia+stem+cells.">Mesenchymal niche-specific expression of cxcl12 controls quiescence of treatment-resistant leukemia stem cells.</a> Cell Stem Cell. 24 (5), 769-784 (2019).</li><li>Duarte, D., 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=Inhibition+of+endosteal+vascular+niche+remodeling+rescues+hematopoietic+stem+cell+loss+in+aml.">Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in aml.</a> Cell Stem Cell. 22 (1), 64-77 (2018).</li><li>O'flanagan, C. 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=Dissociation+of+solid+tumor+tissues+with+cold+active+protease+for+single-cell+rna-seq+minimizes+conserved+collagenase-associated+stress+responses.">Dissociation of solid tumor tissues with cold active protease for single-cell rna-seq minimizes conserved collagenase-associated stress responses.</a> Genome Biol. 20 (1), 210 (2019).</li><li>Stoeckius, 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=Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics.">Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.</a> Genome Biol. 19 (1), 224 (2018).</li></ol>

D.T.S. is a director and shareholder for Agios Therapeutics and Editas Medicines; a founder, director, shareholder, and scientific advisory board member for Magenta Therapeutics and LifeVault Bio, a shareholder and founder of Fate Therapeutics and Garuda Therapeutics, and a director, founder, and shareholder for Clear Creek Bio, a consultant for FOG Pharma, Inzen Therapeutics and VCanBio, and a recipient of sponsored research funding from Sumitomo Dianippon. D.T.S and K.G are inventors of patent US20220143099A1.

Stromal cells in hematopoietic organs are critical for normal blood production and hematopoietic stroma perturbations can result in severe impairments in hematopoietic maintenance and response to stress9,23,24. Insight into hematopoietic stromal cells is essential for understanding hematological diseases7,9,10,24 and for the ability to develop therapeutics to combat them25,26. Reproducibly separating hematopoietic stroma from parenchyma is however a challenge, particularly with the advent of single-cell multiomics which require high-quality sample preparations while maximizing stromal cell yield and variety. The protocols described here have been optimized to be compatible with most downstream multiomics applications while meeting the challenges of isolating hematopoietic stromal cells1,2,19.
The first issue to consider for isolation of hematopoietic stromal cells is dissociation conditions. Optimal dissociation is critical to release stroma cells from the tissue to reflect full heterogeneity of the stroma compartment1,2. Choosing the right dissociation mix depends on the experimental scope and downstream protocols. We would recommend preparing dissociation mixes as freshly as possible and keeping them on ice until use. Some components are hard to dissolve such as dispase and might require more time for&#160;pipet mixing. Vortexing is not recommended as this may disrupt the native conformation of the digestion enzymes, ultimately leading to their inactivation. Importantly, EDTA will inhibit enzymatic activity and should be avoided during active digestion steps, but can be employed to stop the reaction if the enzyme containing buffer cannot be removed promptly. In addition to the enzyme mixture itself, two points are critical to efficient dissociation of tissue, (1) thorough crushing and cutting to increase surface area and, (2) sufficient incubation time with the digestion enzymes. Human thymic tissue is for instance significantly harder to dissociate than murine tissue and digestion times must therefore be longer. If adjustment is not made for human tissue, stromal yield and cell variety will be decreased (Figure 2A). Dissociation enzymes require an optimal temperature of 37 &#176;C which inevitably influences gene expression27. Although cold or lower temperature dissociation would be desirable to conserve cell states, currently there are no commercially available cold-active dissociation enzyme combinations suitable for bone and bone marrow. For any tissue that requires digestion, the heterogeneity and the percent contribution acquired from different cell types will invariably change with the digestion time, enzyme cocktail composition and processing. Downstream of these initial steps, heterogeneity and representation will be further limited by filtration steps such as nozzle sizes during sorting and capture of challenging cell-types (e.g., adipocytes from stroma) and fluidic device channel diameters. Thus, when comparing datasets, we should be mindful that these differences in protocols can impact the representation of subpopulations. It logically follows that we must be vigilant about keeping processing steps equal when measuring the impact of perturbations in stromal systems.
While the cost of sequencing has been and is decreasing, single cell technologies are often limited by the number of cells that can be analyzed. To optimize the capacity of each reagent kit, hematopoietic cell magnetic depletion was employed whenever possible. This enables the avoidance of a high number of contaminant cells which are much more numerous than stroma in both bone marrow and thymic sample fractions. However, if the starting material is limited, as in the case of post-transplant thymic tissue, the increased, non-specific loss of cells that magnetic depletion strategies invariably incur, may make this inadvisable. In addition, during preparation and digestion of the tissues, crushing, cutting and enzymatic release of extracellular matrix components generates a large amount of debris. This type of contaminant, if left in the sample for analysis, can cause clogging issues in downstream processing, cause negative wells for single cell index sorting and highly inflate parent gate percentages in analysis. Calcein staining was used to gate positive for cells/live cells. Calcein comes in different colors which makes it highly adaptable to your staining matrix. When working with calcein be careful to not subject the dye to repeated freeze-thaw cycles (i.e., always prepare fresh and keep at room temperature after dissolving).
The ability to multiplex samples based on antibodies barcoded with DNA oligos28 allows increased utility of single cell sequencing reactions on platforms such as 10x Genomics. The hashtagging antibodies designed for sample multiplexing come with different targets for cell surface molecules or the nuclear membrane and specificity for both mouse and human. While the cell hashtagging system has been used successfully for multiple tissues, experiments combining digestion enzyme mixes with subsequent use of hashtags can lead to drastically reduced recovery. We have attempted to include additional washing steps post-dissociation and added EDTA to inhibit&#160;the activity of the digestion enzymes. However, even with these steps, expect lower hashtag identification than hematopoietic cells. It was found that with enzymatic digestion protocols, the hashtag capture rates were between 30%-70%. This occurs despite the fact that the same antibody clone with a fluorochrome conjugate labeled &#62;90% of stromal cells in the same population. Any experimental design pairing enzymatic digestion of tissue with hashtagging should be piloted and caution taken to ensure enzymatic inhibition.

In the healthy adult, de novo production of blood cells occurs in the bone marrow and the thymus. Stromal cells at these sites are essential for maintenance of hematopoiesis, but stroma constitutes less than 1% of the tissue1,2,3,4. Obtaining pure isolates of hematopoiesis supporting stroma therefore constitutes a significant challenge, particularly for single-cell multiomics that requires expedient processing to obtain samples of high quality. Components of different digestion cocktails may interfere with certain steps in multiomics analysis5,6. The protocols presented here detail the isolation of a wide variety of stromal cells from bone marrow and thymic tissues.
Perturbations of stromal constituents in both bone marrow and thymus result in profound disruption in blood cell development and can result in malignancies7,8,9. Hematopoiesis supporting stroma is damaged following cytotoxic conditioning and bone marrow transplantation, resulting in reduced secretion of cytokines and growth factors that sustain hematopoietic stem and progenitor cells (HSPCs)2,10,11. Furthermore, aging affects bone marrow and thymus stromal cells likely contributing to aged hematopoietic phenotypes. The thymus is the first organ to undergo extensive age-associated involution. Fat and fibrotic tissue start replacing T cell supportive stroma as early as the onset of puberty12,13. In the bone marrow, adipocyte content increases with age and the vascular and endosteal niches are significantly remodeled14,15,16.
To enable study of hematopoiesis supportive stroma across multiple stress states and in the case of the thymus of both human and murine tissue, we have optimized previously published digestion protocols1,2,8,17,18. These protocols ensure efficient and reproducible isolation of cells, and they are compatible with single-cell RNAsequencing (scRNAseq) and other types of multiomics.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25200-072</td><td></td></tr><tr><td>7AAD (7-aminoactinomycin D)</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Anti-Human Lineage Cocktail 3-FITC</td><td>BD Biosciences</td><td>643510</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A9647</td><td></td></tr><tr><td>C57Bl/6 mice</td><td>Jackson</td><td>664</td><td>Males or females, 8-12 weeks old</td></tr><tr><td>Calcein&amp;nbsp;</td><td>Fisher Scientific</td><td>65-0853-78</td><td></td></tr><tr><td>Collagenase IV</td><td>Millipore Sigma</td><td>C5138</td><td></td></tr><tr><td>Corning Sterile Cell Strainers, White, Mesh Size: 70 &amp;micro;m</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>Dispase II</td><td>Thermo Fisher Scientific</td><td>17105041</td><td></td></tr><tr><td>Dnase I Solution</td><td>Thermo Fisher Scientific</td><td>90083</td><td>&amp;nbsp;2500 U/mL</td></tr><tr><td>Easysep mouse streptavidin RapidSpheres Isolation kit</td><td>StemCell Technologies</td><td>19860</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A31605-01</td><td>Qualified One Shot</td></tr><tr><td>Human Fc Block</td><td>BD Biosciences</td><td>564220</td><td></td></tr><tr><td>Liberase TM&amp;nbsp;</td><td>Millipore Sigma</td><td>5401127001</td><td>Research Grade</td></tr><tr><td>Medium 199</td><td>Gibco</td><td>12350</td><td></td></tr><tr><td>Mouse anti-human CD235a-BV77</td><td>BD Biosciences</td><td>740785</td><td></td></tr><tr><td>Mouse anti-human CD31-PE/Dazzle594</td><td>Biolegend</td><td>303130</td><td></td></tr><tr><td>Mouse anti-human CD45-BV77</td><td>Biolegend</td><td>304050</td><td></td></tr><tr><td>Mouse anti-human CD4-BV605</td><td>BD Biosciences</td><td>562658</td><td></td></tr><tr><td>Mouse anti-human CD66b-FITC</td><td>BD Biosciences</td><td>555724</td><td></td></tr><tr><td>Mouse anti-human CD8-APC/Cy7</td><td>BD Biosciences</td><td>557760</td><td></td></tr><tr><td>Mouse anti-human EpCam-BV421</td><td>Biolegend</td><td>324220</td><td></td></tr><tr><td>Protector RNase Inhibitor</td><td>Millipore Sigma</td><td>3335402001</td><td></td></tr><tr><td>Rat anti-mouse CD105-PE /dazzle594</td><td>Biolegend</td><td>120424</td><td></td></tr><tr><td>Rat anti-mouse CD11b-Biotin</td><td>Biolegend</td><td>101204</td><td></td></tr><tr><td>Rat anti-mouse CD140a-APC</td><td>Fisher Scientific</td><td>17-1401-81</td><td></td></tr><tr><td>Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td><td>BD Biosciences</td><td>553142</td><td></td></tr><tr><td>Rat anti-mouse CD31-BUV737</td><td>BD Biosciences</td><td>612802</td><td></td></tr><tr><td>Rat anti-mouse CD31-BV421</td><td>Biolegend</td><td>102424</td><td></td></tr><tr><td>Rat anti-mouse CD3-Biotin</td><td>Biolegend</td><td>100244</td><td></td></tr><tr><td>Rat anti-mouse CD45.2-Biotin</td><td>Biolegend</td><td>109804</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45-PE/Cy7</td><td>Biolegend</td><td>103114</td><td></td></tr><tr><td>Rat anti-mouse CD45R/B220-Biotin</td><td>Biolegend</td><td>103204</td><td></td></tr><tr><td>Rat anti-mouse CD51-PE</td><td>Biolegend</td><td>104106</td><td></td></tr><tr><td>Rat anti-mouse EpCam-BV711</td><td>BD Biosciences</td><td>563134</td><td></td></tr><tr><td>Rat anti-mouse Ly-6A/E(Sca-1)-AF700</td><td>Biolegend</td><td>108142</td><td></td></tr><tr><td>Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin</td><td>Biolegend</td><td>108404</td><td></td></tr><tr><td>Rat anti-mouse Ter119-Biotin</td><td>Biolegend</td><td>116204</td><td></td></tr><tr><td>Rat anti-mouse Ter119-PE</td><td>Biolegend</td><td>116208</td><td></td></tr><tr><td>Rat anti-mouse Ter119-PE/Cy7</td><td>Biolegend</td><td>116222</td><td></td></tr><tr><td>Stemxyme&amp;nbsp;</td><td>Worthington Biochemical</td><td>LS004107</td><td></td></tr></tbody></table>

stromal cell isolation from hematopoietic organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

These protocols yield reproducible stromal cell varieties from the thymus and bone marrow suitable for flow cytometric analysis, as well as single-cell multiomics, such as scRNA sequencing. Murine thymic tissue undergoes significant remodeling in response to stressors, such as the cytotoxic conditioning that precedes bone marrow transplantation or the natural aging process. As a consequence, thymic cellularity is drastically reduced in both of these settings (Figure 1A). While a thymus from an 8-week-old wild-type mouse contains approximately 100 million cells, the cellularity of a 2-year-old mouse can be expected to be half of that, and a thymus 4 days post-irradiation and bone marrow transplantation can be as low as 5,00,000 cells (Figure 1A).
Cytotoxic conditioning will preferentially ablate hematopoietic cells, so the stromal compartment will be proportionally enriched (Figure 1B). However, the overall low cellularity will make isolating sufficient numbers of thymic stromal cells challenging. It may, therefore, be necessary to pool animals to obtain cell numbers compatible with downstream multiomics applications and to minimize the loss of material wherever possible. To perform multiomics on thymic stromal cells, fluorescence-activated cell sorting (FACS) of live, single, CD45-Ter119- cells (Figure 1C) will ensure that sufficient numbers of cells can be sorted from most stress conditions.
To preserve the heterogeneity of the stroma compartment, we typically do not sort on a single positive stroma marker but gate out hematopoietic cells. Including positive stromal cell markers is still recommended for analysis to ensure that proper tissue dissociation has been achieved. However, FACS isolation is imperfect, and as determined by scRNAseq, 30%-50% of the sorted cells will be of hematopoietic origin (Figure 1D,E). This can be improved upon by magnetically depleting CD45+ cells prior to FACS; however, this is not recommended for conditions of reduced thymic cellularity (Figure 1A) as the loss of material that comes with the extra processing involved in magnetic depletion results in stroma yields too low for downstream applications.
For the isolation of human thymic stroma, the most critical step is the enzymatic digestion. If the dissociation step is rushed, the overall stromal cell yield, as well as stromal cell subtype representation, will be severely diminished (Figure 2A). It is also recommended to always include the markers of thymic endothelium and epithelium to ensure that tissue digestion has been optimal, as simply gating negative for hematopoietic markers will not be sufficient to gauge actual stromal cell release. This is exemplified in Figure 2A, where the percentage of cells negative for hematopoietic markers is higher in the sample digested for 30 min compared to the sample incubated for 90 min. However, as seen in the consecutive gate, the yield of endothelial and epithelial cells is very poor in the shorter digestion protocol (Figure 2A).
For FACS isolation of human thymic stromal cells for multiomics applications, staining with a hematopoietic lineage antibody cocktail, as well as CD4 and CD8 antibodies is suggested, in addition to the pan-hematopoietic marker CD45 and the erythroid marker CD235a (Figure 2B) to improve stromal cell enrichment. Thus, for human multiomics analysis of thymic stroma, live, single, CD45-CD235a-Lineage-CD4-CD8- cells should be FACS sorted (Figure 2B). As seen for scRNAseq of murine samples, this still results in contamination of 30%-50% hematopoietic cells, but this still gives acceptable resolution of the human thymus stromal compartment (Figure 2C,D).
For bone and BM stroma, the same strategy was used as for thymic tissue; stromal cells are isolated by gating on cells negative for hematopoietic markers, and further stromal cell markers are included as digestion controls (Figure 3). The dissociation of calcified tissue like bone results in a significant accumulation of debris. The addition of calcein as a viability dye will allow the exclusion of most of this debris, which otherwise falsely inflates sorted numbers and decreases gated population frequencies (Figure 3). This is exemplified in the FACS plots comparing gating on all cells and just gating on Calcein+ (Figure 3). As seen for human thymic stromal cells, the percentage of hematopoietic marker-negative cells is not a reliable readout for bone and BM tissue dissociation efficiency (Figure 3). While the CD45-Ter119- gate can contain an abundance of cells in a sample where the digestion step failed, further analysis of typical bone and BM stromal cell markers CD31, CD140a, and CD51 clearly demonstrate poor stroma release (Figure 3 and Figure 4).
For FACS isolation of murine bone and BM stromal cells, using the gating strategy is suggested as in Figure 3, where we only sort CD45-Ter119-Calcein+ but monitor the forward and side scatter properties and stromal cell marker expression of the sorted population to ensure optimal digestion (Figure 3A). The combination of hematopoietic lineage-positive magnetic cell depletion of the bone marrow prior to FACS and sorting on the shown gates results in single-cell sequencing of cells that are &#62;90% stroma. An example of such a scRNA seq run (Figure 5A,B) illustrates this low hematopoietic contamination after transcriptome-based annotation of the analyzed cells (examples of hematopoietic marker genes Figure 5C). This is in stark contrast to the thymic stromal preparations where magnetic cell depletion was not performed before the sorting step due to the limited starting material in post-transplant thymic tissue (Figure 1F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66231/66231fig01.jpg" /> 
Figure 1: Flow cytometric and single-cell RNA sequencing analysis of murine thymic stroma. (A) Quantification of thymocyte numbers in 8-week-old mice (Control), 8-week-old mice lethally irradiated and transplanted 4 days prior (Transplant) and 2-year-old mice (Aged). (B) Representative flow cytometry plots demonstrating the enrichment in stromal cell subtypes 4 days following lethal irradiation and transplantation. (C) Flow cytometry gating strategy to sort murine thymic stroma for single-cell multiomics analysis. (D) UMAP of murine thymus indicating stroma (blue) and hematopoietic cells (red). (E) UMAPs showing murine hematopoietic marker genes in thymus samples. (F) Quantification of murine stromal and hematopoietic cells in thymus scRNAseq samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66231/66231fig01large.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/66231/66231fig02.jpg" /> 
Figure 2: Flow cytometric and single-cell RNA sequencing analysis of human thymic stroma. (A) FACS plots showing representative results of the impact of different digestion times on the release of human thymic stromal cell populations. (B) Flow cytometry gating strategy to sort human thymic stroma for single-cell multiomics analysis. (C) UMAP of human thymus indicating stroma (blue) and hematopoietic cells (red). (D) UMAPs showing human hematopoietic marker genes in thymus samples. (E) Quantification of human stromal and hematopoietic cells in thymus scRNAseq samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66231/66231fig02large.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/66231/66231fig03.jpg" /> 
Figure 3: Murine bone marrow stromal markers and Calcein are helpful to estimate dissociation efficiency. (A) Representative flow cytometry analysis of CD45-Ter119- murine bone marrow stromal cells gated on all cells and Calcein+ cells. With subsequent CD31- gating showing a higher proportion of CD140a+CD51+ cells when selecting Calcein+ cells. (B) Flow cytometry analysis representative of a failed bone digestion with missing CD140a+CD51+ cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66231/66231fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66231/66231fig04.jpg" /> 
Figure 4: Bone marrow stroma gating strategy. (A) Flow cytometry gating strategy to sort murine bone marrow stroma for single-cell multiomics. (B) Flow cytometry analysis representative of a failed bone marrow digestion with missing CD140a+CD51+ cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66231/66231fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66231/66231fig05.jpg" /> 
Figure 5: Bone marrow single-cell RNA sequencing with stromal and hematopoietic cell composition. (A) UMAP of murine bone and bone marrow indicating stroma (blue) and hematopoietic cells (red). (B) Quantification of stromal and hematopoietic cells in murine bone and bone marrow samples. (C) UMAP of hematopoietic marker genes in murine bone marrow samples. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66231/66231fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stromal Cell Isolation From Hematopoietic Organs at JoVE.com

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

stromal-cell-isolation-from-hematopoietic-organs

Nanyang Technological University

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Immunology and Infection

1.0K Views.  Massachusetts General Hospital. Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Video: Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

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.

Biology

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 of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Bioengineering

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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 ...

Obtaining Single-Cell Suspensions from Murine Brain and Skull Bone Marrow

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...