Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME’s intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells

This protocol is used in: eLife (2023), DOI: 10.7554/eLife.81656 The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HMEforming cells, notably bone marrow stromal cells (BMSCs).The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies.The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis.Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME.By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision.The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations.This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche.

Background
In human bone marrow, hematopoietic stem cells (HSCs) and their progenies are contained in a specialized microenvironment that regulates HSC maintenance and differentiation.Despite the important role of this hematopoietic environment (HME), its cellular composition, potential heterogeneity, and cellular hierarchy remain poorly defined.This is mainly due to the extremely low frequency of the HME-forming cells, the so-called bone marrow stromal (stem) cells, BMSCs [1,2].The conventional approach to studying the human bone marrow microenvironment is mainly based on the analysis of different cell types defined by the expression of a limited number of known surface markers, which results in an underestimation of cellular complexity.Novel single-cell-based omics approaches, on the other hand, have the potential to provide detailed insights into complex cellular organization and function.However, whereas bulk preparations of bone marrow cells allow for analysis of the majority of cells, important low-frequency cell populations such as BMSCs will escape detailed analysis.Therefore, we developed a strategy to combine singlecell RNA sequencing of sorted non-hematopoietic BM cells with highly enriched BMSCs to resolve the cellular heterogeneity of the human bone marrow microenvironment at the highest possible resolution based on transcriptomic profiling [3].Our approach is based on the expression of CD45, CD235a, and CD271.CD45 is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene (protein tyrosine phosphatase receptor type C).CD45 is considered a panhematopoietic marker and is widely used to select all hematopoietic cells and precursors except erythroid cells [4].CD235a, also known as glycophorin A (GYPA), is a major intrinsic membrane protein of erythrocytes and a distinct marker of erythroblasts [5].Therefore, we chose to use the combination of CD45 and CD235a to enrich nonhematopoietic human bone marrow microenvironment cells based on their low or absent expression of both CD45 and CD235a.Finally, BMSCs were highly enriched by sorting CD45 low/-CD235a -/CD271 + cells, which is based on data by us and others demonstrating that the CD271 positive BM cell population contains all assayable stromal cells [5][6][7].This paper describes a step-by-step protocol to isolate cells from the human bone marrow microenvironment for single-cell RNA sequencing [3] that can certainly be applied to other state-of-the-art omics approaches.Thus, this protocol contributes valuable information that, when combined with future research efforts, will contribute to a deeper understanding of the intricate cellular interactions within the bone marrow niche.2. Transfer the bone marrow aspirate to a sterile T-75 culture flask.
3. Add 100 mL of Ficoll buffer to the flask and mix by pipetting up and down.4. Carefully layer 30 mL of bone marrow-Ficoll buffer mix over Ficoll-Paque Premium in each 50 mL Falcon tube. 5. Centrifuge at 300× g for 30 min at room temperature without breaks [acceleration rate: 6 (maximum 10); deceleration rate: 0] utilizing a centrifuge equipped with a swinging bucket rotor.6. Collect interphases containing mononuclear cells (Figure 1) into five new 50 mL Falcon tubes and fill up with Ficoll buffer to 50 mL.2. Set up a gate to remove FSC-low populations by drawing a polygonal gate around the regions containing FSC-medium and FSC-high particles, as the FSC-low population consists of cell debris, air bubbles, and laser noise (see Figure 2A).3. Create a Forward Scatter-Height (FSC-H) versus FSC-A plot and exclude doublets and multiplets by gating out cells with higher area signal values (FSC-A) (Figure 2B). 4. Create a FSC-A versus DAPI plot to exclude non-viable cells by gating out the DAPI-high cells (Figure 2C). 5. Create a FITC versus PE-Cy5 plot to exclude CD45-high and CD235-expressing cells (Figure 2D).6. Create a FSC-A versus APC plot to exclude CD271 negative cells (Figure 2E). 7. Sort 100 events from the CD45 low/-CD235a -or CD45 low/-CD235a -CD271 + cell fractions into a tube containing 100 µL of ice-cold Collection buffer.

Data analysis
To evaluate the effectiveness of cell isolation, various parameters were assessed, including (1) the efficiency of Ficoll-Paque gradient separation, (2) the viability of bone marrow mononuclear cells, (3) the real-time gating strategy utilizing BD FACS Diva, and (4) the efficiency of cell sorting.
1. Ficoll-Paque gradient separation After centrifugation, observe the tube to identify different layers.Typically, layers include plasma, a mononuclear cell layer (buffy coat), a Ficoll-Paque layer, and a red blood cell layer.A well-performed Ficoll-Paque gradient separation should result in four distinct layers with the mononuclear cell layer containing a high concentration of nucleated cells.The efficiency of separation can be evaluated by the presence of a clear interface between layers and minimal contamination between different cell populations.

Cell viability
The quality of isolated mononuclear cells can be evaluated using various viability assays.A cell viability of over 90% is recommended, particularly for cell sorting procedures.Cell viability is calculated using the following formula: Viability (%) = (1 -Total number of stained cells / Total number of cells) × 100

Gating strategy
An effective real-time gating strategy is paramount for the precise collection of high-quality, viable nonhematopoietic cells for single-cell RNA sequencing.As illustrated in Figure 2, the gating strategy employed ensured the targeted isolation of the desired cell population while meticulously excluding debris (Figure 2A), doublets (Figure 2B), non-viable or damaged cells (Figure 2C), and unwanted hematopoietic cells (Figure 2D).The sequential application of these gates progressively refined the cell population, culminating in the final selection of CD271-positive cells (Figure 2E) for subsequent downstream analyses.

Sorting efficiency
After the initial sorting process, a critical step in ensuring data integrity is the reanalysis of sorted cells.This involves a reassessment of the sorted cell population using the same flow cytometry gating strategy.By performing sorting reanalysis, we could evaluate the sorting purity and efficiency, identify contaminants and refine the gating strategy if necessary.Through meticulous verification and validation, researchers can ensure the consistency and accuracy of sorted cell samples, facilitating trustworthy results in subsequent experiments and analyses.It is advisable to aim for a sorting purity exceeding 85% for optimal suitability in subsequent analyses.

Validation of protocol
This protocol or parts of it has been used and validated in the following research article: Li et al. [3].Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing.eLife (Figure 1, panel A).

Figure 1 . 7 .
Figure 1.Ficoll-Paque separation demonstration and expected layers after density gradient centrifugation.Each layer represents components with different densities, allowing for the separation and isolation of specific cell populations from the bone marrow aspirates.Cell separation layers starting from the top: plasma; interphase with mononuclear cells; Ficoll-Paque; and bottom fraction with leucocytes,

6 Published: Jun 20, 2024 4 . 6 .C. FACS sorting 1 .
Cite as: Li, H. et al. (2024).Isolation of Human Bone Marrow Non-hematopoieticCells for Single-cell RNA Sequencing.Bio-protocol 14(12): e5020.DOI: 10.21769/BioProtoc.5020.Wash the stained cells by adding 1 mL of Sorting buffer to the tubes and centrifuge tubes for 5 min at 800× g at 4 °C. 5. Resuspend Tubes a-e (a'-g' for panel 2) with 500 μL of Sorting buffer and add 2.5 μL of sterile DAPI stock.Resuspend Tube f (h' for panel 2) in 3 mL of Sorting buffer and add 15 μL of DAPI stock.7. Pass the cells through a 30 μm Filcon or any equivalent strainer.8. Proceed quickly with FACS sorting.Open the BD FACSDiva Software in the software interface and locate the workspace where you can create plots.Create a plot and choose the parameters to make a Forward Scatter-Area (FSC-A) versus Side Scatter-Area (SSC-A) plot.Adjust the individual FSC and SSC photomultiplier tube settings to visualize the expected cell populations (Figure 2A).

1 .
Incubate cells in Blocking buffer for 20 min at room temperature.2.Aliquot the cells and antibodies according to Table1to isolate CD45 low/-CD235a -cells.Aliquot the cells and antibodies according to Table2to isolate CD45