Stromal Cell Isolation From Hematopoietic Organs

Summary

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.

Abstract

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.

Introduction

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 tissue^1,2,3,4. 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 analysis^5,6. 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 malignancies^7,8,9. 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 puberty^12,13. In the bone marrow, adipocyte content increases with age and the vascular and endosteal niches are significantly remodeled^14,15,16.

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 protocols^1,2,8,17,18. These protocols ensure efficient and reproducible isolation of cells, and they are compatible with single-cell RNAsequencing (scRNAseq) and other types of multiomics.

Protocol

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

1. Prepare the following buffers and solutions.
   1. Medium 199 (M199) with 2% (v/v) fetal bovine serum (FBS) (see Table of Materials).
   2. Murine dissociation cocktail: 0.5 WU/mL Liberase TM and 6.3 U/mL DNase I in M199 + 2% FBS (see Table of Materials).
   3. M199 with 2% (w/v) Bovine serum albumin (BSA).
      NOTE: FBS containing medium can be switched to BSA to improve the RNA quality.
2. Perform the murine thymus dissection
   1. 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.
   2. 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.
      1. 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.
   3. Using a dissection microscope, remove any extrathymic tissue using blunt-ended forceps and micro-spring scissors.
3. Perform enzymatic dissociation of murine thymic tissue (30 min in increments of 10 min)
   1. 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.
   2. 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.
   3. To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 °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 °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.
   4. Place the 15 mL tube in a rack and let the tissue pieces settle. Carefully remove the supernatant and pass it over a 70 µm cell strainer into an ice-cold 50 ml tube.
   5. 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.
   6. Add 20 mL of M199 + 2% FBS to the collected cell suspension. Spin down at 500 x g for 5 min at 4 °C.
   7. 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.
   8. 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.
4. Perform fluorescence activated cell sorting of murine thymic stroma
   1. Spin down the cells at 500 x g for 5 min at 4 °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).
   2. Add murine Fc Block (see Table of Materials) at a concentration of 2 µg/mL and incubate for 10 min at 4 °C.
   3. Stain the cells with the following antibodies: CD45-PE/Cy7 (4 µg/mL), Ter119-PE (4 µg/mL), CD31-BUV737 (2 µg/mL), and EpCam-BV711 (2 µg/mL) (see Table of Materials) for 30 min at 4 °C.
   4. Wash the samples in M199 + 2% BSA and spin down at 500 x g for 5 min at 4 °C.
   5. Remove supernatant and resuspend cells in M199 + 2% BSA containing a viability dye such as DAPI (0.5 µg/mL) or 7-AAD (1 µg/mL) (see Table of Materials). Proceed to FACS isolation of thymic stromal cells following established protocols¹⁹.

2. Preparation of human thymic tissue

1. Prepare the following buffers and solutions.
   1. Medium 199 (M199) with 2% (v/v) FBS.
   2. M199 with 1.5% (w/v) BSA.
   3. 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.
2. Perform human thymic tissue isolation
   1. 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.
   2. 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.
   3. 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.
   4. 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.
3. Perform enzymatic dissociation of human thymic tissue (90 min in increments of 30 min)
   1. 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.
   2. 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.
   3. 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 °C for 30 min.
   4. 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 µm cell strainer into a new 50 ml tube placed on ice.
   5. Repeat steps 2.3.2-1.3.4 for a total of two rounds of digestion with the human dissociation cocktail.
   6. 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 °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.
   7. 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 µm cell strainer. Add 3 mL of FBS to the sample to break the trypsin reaction and place on ice.
   8. Proceed to counting the cells as described in step 1.3.
4. Perform fluorescence activated cell sorting of human thymic stroma
   1. 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.
   2. Incubate with human Fc Block (5 µg/mL) (see Table of Materials) for 10 min at 4 °C.
   3. Stain the sample for 30 min at 4 °C with CD45-BV711 (2.5 µg/mL), CD235a-BV711 (2.5 µg/mL), Lineage-cocktail-FITC (3.76 µg/mL), CD66b-FITC (20 µL/100 µL sample), CD8-APC/Cy7 (5 µL/100 µL sample), CD4-BV605 (5 µL/100 µL sample), CD31-PE/Dazzle594 (10 µg/mL), and EpCam-BV421 (2.5 µg/mL) (see Table of Materials).
   4. Wash the samples in M199 + 1.5% BSA and spin down at 500 x g for 5 min at 4 °C.
   5. Remove the supernatant and resuspend cells in M199 + 1.5% BSA containing a viability dye such as DAPI (0.5 µg/mL) or 7-AAD (1 µg/mL). Proceed to FACS isolation of thymic stromal cells following established protocols¹⁹.

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.

1. Prepare the following buffers and solutions.
   1. M199 with 2% (v/v) FBS.
   2. Murine B&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&M dissociation mix fresh and keep mix on ice until use.
   3. M199 with 0.5% (w/v) BSA.
   4. Phosphate-buffered saline (PBS) with 0.5% (w/v) BSA.
   5. 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.
2. Perform murine bone marrow separation
   1. Euthanize the mouse by CO₂ asphyxiation (following institutionally approved protocols). Spray with 70% ethanol to wet the fur.
   2. Carefully remove the femur, tibia, pelvis, and humerus from each side and place them in a well containing M199 + 2% FBS on ice^21,22.
   3. 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.
   4. 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.
   5. 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.
3. Perform enzymatic dissociation of murine bone marrow tissue (total 30 min in increments of 10 min)
   1. 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.
   2. Add 4 mL of B&M dissociation cocktail to the 15 mL tube and place it in a 37 °C water bath (no shaking) for 10 min.
   3. After 5 min, invert the tube three times and place it back in the water bath.
   4. After the first 10 min, ensure the pellet is at the bottom and collect the supernatant while filtering through a 70 µm cell strainer into an ice-cold 50 mL tube. Add 2 mL of fresh B&M dissociation cocktail to the remaining BM pellet and place it back in the water bath.
   5. 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.
4. Perform hematopoietic cell depletion of murine bone marrow tissue.
   1. Centrifuge cells at 500 x g for 5 min at 4 °C. Resuspend in 500 µL 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.
   2. Incubate on an orbital shaker for 10 min at room temperature in the dark.
   3. Vortex the magnetic beads thoroughly and add 25 µL of magnetic beads to the tube.
   4. Incubate on an orbital shaker for 5 min at room temperature in the dark.
   5. 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.
5. Perform enzymatic dissociation of murine bone tissue.
   1. 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 µm 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.
   2. 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.
   3. Place the bone fraction in 5 mL of murine B&M dissociation (see Table of Materials) mix in a new 50 mL tube.
   4. To prevent leakage, wrap the cap of the tube in paraffin film. Then place the tube horizontally in a 37 °C water bath with 120 rpm shaking for 30 min.
   5. 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 µm 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.
6. Perform fluorescence activated cell sorting of murine bone and bone marrow stroma.
   1. Spin down the cells at 500 x g for 5 min at 4 °C.
   2. Resuspend in murine Fc Block at a concentration of 10 µg/mL in PBS + 0.5% BSA + 2 mM EDTA and incubate for 10 min at 4 °C.
   3. Stain the cells (up to 10 million cells/100 µL total staining volume) with the following antibodies: CD45-PE/Cy7 (1 µg/mL), Ter119-PE/Cy7 (1 µg/mL), CD31-BV421 (0.66 µg/mL), CD140a-APC (2 µg/mL), Sca1-AF700 (0.66 µg/mL), CD51-PE (2 µg/mL), CD105-PE/dazzle (2 µg/mL) (see Table of Materials).
   4. Leave Calcein-AM to equilibrate to room temperature and resuspend in 50 µL 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 µg/mL) already resuspended in the antibody staining cocktail and incubate for 20-30 min at 4 °C in the dark.
   5. Wash the samples in PBS + 0.5% BSA + 2 mM EDTA and centrifuge at 500 x g for 5 min at 4 °C.
   6. 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.

Results

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 ...

Discussion

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 stress^9,23,24. Insight into hematopoietic stromal cells is essential for understanding hematological diseases^7,9,10,

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Thermo Fisher Scientific	25200-072
7AAD (7-aminoactinomycin D)	BD Biosciences	559925
Anti-Human Lineage Cocktail 3-FITC	BD Biosciences	643510
Bovine Serum Albumin	Millipore Sigma	A9647
C57Bl/6 mice	Jackson	664	Males or females, 8-12 weeks old
Calcein	Fisher Scientific	65-0853-78
Collagenase IV	Millipore Sigma	C5138
Corning Sterile Cell Strainers, White, Mesh Size: 70 µm	Fisher Scientific	08-771-2
DAPI (4',6-Diamidino-2-Phenylindole, Dilactate)	Biolegend	422801
Dispase II	Thermo Fisher Scientific	17105041
Dnase I Solution	Thermo Fisher Scientific	90083	2500 U/mL
Easysep mouse streptavidin RapidSpheres Isolation kit	StemCell Technologies	19860
Fetal Bovine Serum	Gibco	A31605-01	Qualified One Shot
Human Fc Block	BD Biosciences	564220
Liberase TM	Millipore Sigma	5401127001	Research Grade
Medium 199	Gibco	12350
Mouse anti-human CD235a-BV77	BD Biosciences	740785
Mouse anti-human CD31-PE/Dazzle594	Biolegend	303130
Mouse anti-human CD45-BV77	Biolegend	304050
Mouse anti-human CD4-BV605	BD Biosciences	562658
Mouse anti-human CD66b-FITC	BD Biosciences	555724
Mouse anti-human CD8-APC/Cy7	BD Biosciences	557760
Mouse anti-human EpCam-BV421	Biolegend	324220
Protector RNase Inhibitor	Millipore Sigma	3335402001
Rat anti-mouse CD105-PE /dazzle594	Biolegend	120424
Rat anti-mouse CD11b-Biotin	Biolegend	101204
Rat anti-mouse CD140a-APC	Fisher Scientific	17-1401-81
Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Biosciences	553142
Rat anti-mouse CD31-BUV737	BD Biosciences	612802
Rat anti-mouse CD31-BV421	Biolegend	102424
Rat anti-mouse CD3-Biotin	Biolegend	100244
Rat anti-mouse CD45.2-Biotin	Biolegend	109804
Rat anti-mouse CD45-PE/Cy7	Biolegend	103114
Rat anti-mouse CD45-PE/Cy7	Biolegend	103114
Rat anti-mouse CD45R/B220-Biotin	Biolegend	103204
Rat anti-mouse CD51-PE	Biolegend	104106
Rat anti-mouse EpCam-BV711	BD Biosciences	563134
Rat anti-mouse Ly-6A/E(Sca-1)-AF700	Biolegend	108142
Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin	Biolegend	108404
Rat anti-mouse Ter119-Biotin	Biolegend	116204
Rat anti-mouse Ter119-PE	Biolegend	116208
Rat anti-mouse Ter119-PE/Cy7	Biolegend	116222
Stemxyme	Worthington Biochemical	LS004107