stromal cell extraction from mouse bone and bone marrow for superior single cell multi&#45;omics data

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

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.

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

Stromal Cell Isolation From Hematopoietic Organs

Development of hematic cells in bone marrow and thymus is highly dependent on stromal cells for support. Perturbations of hematopoietic stromal cell function can lead to inefficient or even malignant blood production. Our lab is focused on understanding stromal cell regulation of hematopoiesis and how it can be manipulated for therapeutic benefits.The advent of single cell sequencing has driven a cascade of recent advances in many research fields. This is also the case in our field where single cell resolution has enabled the identification of heterogeneous stromal cell populations in hematopoietic organs that were previously unknown. Our protocols are optimized to address this, and we hope they will enable more research on stromal cells to tackle critical questions in the field.

Stromal Cell Extraction from Mouse Bone and Bone Marrow for Superior Single Cell Multi&#45;Omics Data

To begin, take the femur, tibia, pelvis, and humerus bones isolated from a mouse. Use a scalpel to cut through the growth plates of long bones, removing the epiphysis. Place it in a 10-centimeter dish with M199 medium plus 2%FBS on ice.Next, use a 28-gauge needle and a 10-milliliter syringe filled with M199 medium plus 2%FBS to flush the marrow into a 15-milliliter tube on ice. Place the flushed bone in the dish with the epiphysis. Leave the 15-milliliter tube upright on ice until the marrow settles.Then remove the M199 medium plus 2%FBS Add four milliliters of the B&M dissociation cocktail to the 15-milliliter tube and incubate in a 37 degree Celsius water bath. After incubation, collect the supernatant and filter through a 70-micrometer cell strainer into a cold 50-milliliter tube. Add two milliliters of fresh B&M dissociation cocktail to the remaining bone marrow pellet and return it to the water bath.Using a one-milliliter pipette, vigorously pipette any remaining bone marrow pieces. Collect all cell suspension in the same tube as before. Add 20 milliliters of M199 medium with 0.5%BSA, and two-millimolar EDTA to the cell suspension.Afterward, centrifuge the cells at 500 G for five minutes at four degrees Celsius. Resuspend the pellet in 500 microliters of PBS with a 0.5%BSA and biotin antibodies. Incubate on an orbital shaker for 10 minutes at room temperature in the dark.Next, vortex the magnetic beads thoroughly. Add 25 microliters of the beads to the tube and incubate again on the orbital shaker. Place the tube in a matching magnet for two to three minutes and collect the supernatant in a fresh tube.Break the bones into smaller pieces in the 10-centimeter dish using the flat cap end of a 50-milliliter tube. Filter the supernatant through a 70-micrometer cell strainer to isolate bone fragments. Cut the bone fragments in the cell strainer with scissors.Place the filter with the bone fragments on a 50-milliliter tube and open the filter with the scalpel. With tweezers, transfer the bone fraction to five milliliters of murine B&M dissociation mix. Then place the tube horizontally in a 37 degree Celsius water bath with 120 RPM shaking for 30 minutes.After digestion, add 25 milliliters of M199 medium with 0.5%BSA and two-millimolar EDTA. Filter the supernatant containing stromal cells through a 70-micron cell strainer into a cold 50-milliliter tube.

Research

JoVE Journal

Immunology and Infection

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