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. Author manuscript; available in PMC: 2022 Aug 29.
Published in final edited form as: Methods Mol Biol. 2021;2346:21–34. doi: 10.1007/7651_2020_298

Interactions of Hematopoietic Stem Cells with Bone Marrow Niche

Xinghui Zhao 1, Cuiping Zhang 1, Xiaojing Cui 1, Ying Liang 1
PMCID: PMC9423787  NIHMSID: NIHMS1828403  PMID: 32504296

Abstract

Hematopoietic stem cells (HSCs) are ultimately responsible for the lifelong renewal of all blood cell lineages. In the bone marrow (BM), HSCs reside in specialized microenvironments referred to as the “niche.” HSC niche consists of complex components including heterogeneous cell populations, growth factors, and extracellular matrix molecules. The crosstalk between HSCs and their niche is essential to regulate the survival, self-renewal, migration, quiescence, and differentiation of HSCs. The application of mice models with endogenous ablation of specific cell types, advanced imaging technologies, high-throughput single-cell RNA sequencing, and single-cell mass cytometry methods have provided deep insights into communications between HSCs and niche cells. In this chapter, we have focused on three important cell types in the BM niche: mesenchymal stem cells (MSCs), osteoblasts (OBs), and endothelial cells (ECs). In order to address the interaction between HSCs and these three cell populations in BM niche, we have described methodology for (1) collecting total BM from femur and tibia of C57BL/6 mice; (2) analyzing or sorting of MSCs, OBs, and ECs based on the selection of surface markers CD45, Ter119, CD31, Sca1, and CD51 with flow cytometry; and (3) co-culturing the sorted cells with purified HSCs for further functional assays of HSCs.

Keywords: Endothelial cells (ECs), Hematopoietic stem cells (HSCs), Mesenchymal stem cells/mesenchymal stromal cells (MSCs), Osteoblasts (OBs)

1. Introduction

Hematopoietic stem cells (HSCs) are characterized by their long-term self-renewal capacity and pluripotency [1]. HSC is a heterogeneous population consisting of a long-term subset (LT-HSCs), a short-term subset (ST-HSCs), and multipotent progenitors (MPPs). LT-HSCs are capable of indefinite self-renewal to sustain HSC pool and can differentiate into ST-HSCs and MPPs. ST-HSCs have limited self-renewal capacity, whereas MPPs lose this capacity with full differentiation into lineage-specific progenitor cells. Common lymphoid progenitor (CLP) cells produce lymphoid cells, and common myeloid progenitor (CMP) cells give rise to myeloid cells [2]. HSC population heterogeneity can also be evaluated by its lineage differentiation potential; that is, myeloid-biased HSCs, lymphoid-biased HSCs, and balanced HSCs. HSC hierarchy and heterogeneity are observed in both human and mouse hematopoietic systems [3–5]. In adults, HSCs locate primarily within the BM, which is a complex organ containing many different cell types [6]. HSC activities such as self-renewal and differentiation are tightly regulated by cell-intrinsic factors as well as cell-extrinsic regulators [7, 8]. Raymond Schofield first introduced the concept of stem cell “niche” in 1978, suggesting that the fate of stem cells is determined by their interaction with the microenvironment they reside in [9, 10].

HSC niche is composed of different cell types and extracellular elements [11, 12]. Now, an increasing number of cell types have been demonstrated to communicate with HSCs and regulate their survival, self-renewal, migration, quiescence, and differentiation [6, 12, 13]. According to the anatomical location, at least two different hematopoietic microenvironments exist in the BM: the endosteal niche and the perivascular niche [14, 15]. The endosteal niche is localized in the outer edge of the BM and includes mesenchymal stem cells (MSCs), osteoblasts, fibroblasts, macrophages, and adipocytes [16–18]. The perivascular (more specifically, arteriolar and sinusoidal) niche is adjacent to the BM vasculature and mainly composed of vascular endothelial cells (ECs) and MSCs [7, 14, 19].

Genetically modified animal models enabling endogenous ablation of various cell types or lineage tracing, combined with reporters and advanced imaging technologies, have helped unravel the communications between HSCs and the niche cells [7, 20–22]. For example, whole-mount tridimensional (3D) BM imaging and computational modeling revealed that quiescent HSCs associate specifically with small arterioles ensheathed by NG2+ (a pericyte marker) pericytes, suggesting that pericytes surrounding arterioles are important for maintaining HSC quiescence in BM [23]. Similar 3D confocal immunofluorescence analysis has also been used on Vwf-GFP (green fluorescence protein) mice, a mice model with green fluorescent protein-labeled myeloid-biased HSCs upon transplantation, to study HSC-niche communications. The results demonstrated that myeloid-biased HSCs are highly enriched in megakaryocytes niche while lymphoid-biased HSCs are preferentially located in arteriolar niche [24]. By crossing Mds1GFP/+ mice in which GFP is highly expressed in HSCs and progenitor cells with Flt3-Cre mice, Christodoulou et al. developed an HSC-specific reporter model which enables direct visualization of long-term HSCs in their native BM microenvironment in live animals without transplantation. They found that HSC expansion is restricted in the BM cavities where bone remodeling occurs, suggesting the previously unknown heterogeneity in HSC niche [25].

Recently, high-throughput single-cell RNA sequencing and single-cell mass cytometry methods helped define discrete cell populations in HSC niches at single-cell resolution and provided insights into the HSCs and niche cells communications [26–30]. Tikhonova et al. profiled 17,374 single cells of the BM niche and mapped the detailed transcriptional landscape of distinct vascular, perivascular, and osteo-lineage components in both homeostasis and stress state. This study revealed that the absence of Dll4 in vascular ECs led to a significant myeloid-biased skewing of HSCs [27]. Baryawno et al. revealed a taxonomy of 17 distinct cell types in BM stroma at the transcriptome level in both normal hematopoiesis and leukemia pathologic state. The study has revealed new groups of mesenchymal, pericyte, fibroblast, and endothelia subpopulations and defined differences among Lepr-, Nestin-, and NG2-expressing cell populations in niche [28]. Using mass cytometry (cytometry by time of flight), Severe et al. defined 28 subsets of BM stromal cells (BMSCs) based on the protein level and found CD73+ subpopulations contribute to HSC engraftment and acute hematopoietic recovery [31]. As technology develops, so does our understanding of BM niche components at both cellular and molecular levels. Significant advances in technology and in our understanding indicate the complexity of niche and its regulation of HSC function in physiological, stress, and malignant conditions.

MSCs, also known as mesenchymal stromal cells or medicinal signaling cells [32], are multipotent cells capable of differentiating into various cell types including osteocytes, chondrocytes, and adipocytes [33, 34]. MSCs are a critical component in HSC niche [7, 35, 36]. It is shown that MSCs can facilitate HSC engraftment in both animal transplantation models and clinical studies [37]. Mesenchymal stem and progenitor cells derived from human BM stroma (CD146+) [38] and mouse fetal bones (CD105+CD51+) [39] are both capable of generating niches containing host-derived HSCs after transplantations. By conjugating nestin, a MSC marker, with GFP, it was discovered that HSCs are closely associated with Nes-GFP+ MSCs in vivo [40]. MSCs are also known as one of the major sources of niche regulatory factors [7, 41]. Genes important to HSC maintenance, such as Cxcl12, stem cell factor (SCF), angiopoietin-1 (ANG-1), interleukin-7 (IL-7), and vascular cell adhesion molecule 1 (VCAM1), are significantly enriched in Nes-GFP+ MSCs than other types of stromal cells [40]. Using mice models with Cxcl12 deletion in different niche cell populations, Greenbaum et al. found that Cxcl12 in early Nestin−LepR− mesenchymal progenitors is required for HSC maintenance [42].

Spatial distribution of transplanted HSCs/hematopoietic progenitor cells (HPCs) shows that they are preferentially located to the endosteal regions [43]. Within the endosteal region, LT-HSCs are much more closer to endosteum and OBs compared with more mature subsets [44]. Osteoblasts are the major cellular component in the endosteal niche and also the first cell population linked to the regulation of HSCs [7]. In mice models where osteoblast cells were specifically ablated in the BM, LT-HSCs demonstrated loss of quiescence and reduced long-term engraftment and self-renewal capacity [45]. Taichman et al. demonstrated that osteoblasts constitutively produce granulocyte colony-stimulating factor (G-CSF) to support human CD34+ cell expansion in vitro [46]. In addition, osteoblasts can also produce other molecules which are important for HSC lodging and maintenance, including osteopontin (OPN), thrombopoietin, annexin-2, and angiopoietin-1 [7, 47]. OPN, a phosphorylated matrix glycoprotein, is an important component within the endosteal niche and functions as a negative regulator of HSC proliferation [48, 49]. OPN-deficient (Opn−/−) mice have an increased BM HSC pools in vivo, highlighting OPN as a potent negative element of the HSC niche [48, 50]. In young mice, transplantation of wild-type LSK cells into Opn−/− recipient mice results in defective engraftment of HSCs within the endosteal region [48]. In aged BM stroma, the expression of OPN is reduced, which contributes to HSC aging [51].

HSCs are intimately associated with ECs throughout their lifecycles from embryonic development to adult and aging [52–54]. ECs are specialized cells lining the interior surface of blood vessels [54]. Rather than acting as an inert barrier, ECs play key roles in maintenance, self-renewal, and differentiation of HSCs. Co-culturing human BM-derived ECs with HSCs and progenitor-enriched CD34+ umbilical cord blood cells showed that BM ECs support long-term hematopoiesis in vitro by elaboration of lineage-specific cytokines [55]. Selective deletion of gp130 in murine hematopoietic cells and ECs led to anemia and myeloid leukocytosis; this defect appeared to reside in the BM microenvironment rather than in the hematopoietic cells themselves. These studies suggest that ECs make an important contribution to hematopoiesis in vivo [56]. Further in vivo studies revealed that specific deletion of Scf [57], Cxcl12 [58], or Jag1 [59] in ECs with Tie2-Cre or VE-cadherin-Cre mice strains impairs HSC maintenance at steady state, confirming the role of ECs as an important source of extracellular factors in HSC niche [7].

In summary, these studies further emphasize the complexity of cellular network in BM niche (Fig. 1). In this chapter, we will focus on the separation and characterization of MSCs, osteoblasts, and ECs in mice BM niche, and their co-culture with HSCs (Fig. 2).

Fig. 1.

Fig. 1

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Distinct cell populations in the BM niche of hematopoietic stem cells (HSCs). HSC niche provides a microenvironment composed of different cell types and extracellular elements. The crosstalk between HSCs and their niche is essential to regulate the survival, self-renewal, migration, quiescence, and differentiation of HSCs. According to the anatomical location, at least two different hematopoietic microenvironments exist in the BM: the endosteal niche and the perivascular niche. Here we show the three important cell types in the BM niche: mesenchymal stem cells/mesenchymal stromal cells (MSCs), osteoblasts (OBs), and endothelial cells (ECs). CXCL12 and stem cell factor (SCF) are two key factors for HSC maintenance secreted mainly by ECs and MSCs. Osteopontin (OPN) and angiopoietin-1 (ANG-1) are two important osteoblast-derived cytokines for lodgement and maintenance of HSCs

Fig. 2.

Fig. 2

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Flowchart for analysis or sorting of mouse osteoblasts, MSCs, and endothelial cells by flow cytometry

2. Materials

2.1. Tissues

Cells are obtained from the long bones of adult C57BL/6 mice (8–12 weeks old).

2.2. Reagents and Supplies

  1. Medium: Hank’s Balanced Salt Solution (HBSS) with 2% fetal bovine serum (FBS). Dulbecco’s Modified Eagle’s medium, 4.5 g/l glucose + L-glutamine + pyruvate (DMEM). Co-culture medium: 1:1 mix of DMEM and α-modified Eagle medium containing 10% FBS, 1% penicillin/streptomycin, and 50 mM 2-mercaptoethanol.

  2. Red cell lysis buffer (10×) (BD Pharmingen™, Catalog No. 555899), diluted with distilled H2O to 1×, and then filter-sterilize through 0.22 μm filter.

  3. Collagenase II solution: 2 mg/ml collagenase II (≥125 U/mg; Catalog No. 17101015, Thermo Fisher Scientific) in DMEM. Make fresh and filter to sterilize.

  4. 70%(v/v) ethanol.

  5. Mouse Recombinant SCF (Catalog No. 78064), Mouse Recombinant Flt3/Flk-2 Ligand (Catalog No. 78011), Mouse Recombinant IL-11 (Catalog No. 78026). All are from Stem Cell Technologies.

  6. Scalpels (no. 10), scissors, tweezers, curved forceps.

  7. Mortar and pestle.

  8. 100 μm cell strainer (VWR, Catalog No. 10054-458).

  9. 1.5 ml microcentrifuge tube, 15 or 50 ml conical tubes.

  10. 5 ml polystyrene round-bottom tube (FACS tube).

  11. 100 mm cell culture petri dish.

  12. Tissue culture-treated 96-well plates.

2.3. Fluorescence-Conjugated Antibodies

  1. APC Rat Anti-Mouse CD45 (BD Pharmingen™, Catalog No. 559864).

  2. APC Rat Anti-Mouse TER-119/Erythroid Cells (BD Pharmingen™, Catalog No. 557909).

  3. Pacific Blue™ anti-mouse CD31 Antibody (Biolegend, Catalog No.102422).

  4. Ly-6A/E (Sca-1) Monoclonal Antibody (D7), PE-Cyanine7 (eBioscience™, Catalog No. 25-5981-82).

  5. PE Rat Anti-Mouse CD51 (BD Pharmingen™, Catalog No. 551187).

  6. 7-AAD (BD Pharmingen™, Catalog No.559925).

2.4. Equipment

  1. Hood for cell culture with vertical laminar flow and equipped with UV light for decontamination.

  2. Centrifuge.

  3. Shaking water bath with temperature control.

  4. Hemocytometer.

  5. Optical microscope.

  6. Flow cytometry analyzer, FACS BD™ LSR II; sorter, FACS Aria™ II.

  7. Incubator.

3. Methods

3.1. Preparing Single-Cell Suspension from Long Bones (Femur and Tibia)

  1. Euthanize the mouse by CO2 asphyxiation, and confirm death by cervical dislocation.

  2. Wet the pelt thoroughly with 70% ethanol. Put the mouse on the sterilized tissue towel (see Note 1).

  3. Make an incision on the back skin using scissors and tweezers. The whole skin is then removed from the cutting site on the back toward the foot. One hand holds the tail, and another hand holds the foot and pulls the leg toward the head to remove the entire intact leg (iliac crest, femur, and tibia) from the animal. Repeat for the remaining legs. Use a scalpel to cut through the tibia just above the ankle to remove the foot (see Note 1).

  4. Position the leg to make the iliac crest facing up, and holding the femur near the knee, scrape the scalpel down the femur toward the iliac crest to dislocate the hip joint. Cut the knee joint in the center. Using a scalpel, scrape bones thoroughly to remove muscles and excess soft tissue. Ensure that the bones are cleaned thoroughly without remaining connective tissue attached. Place the long bone (two sets of tibias and femurs per mouse) in a 15 ml tube with 5 ml HBSS +2% FBS. Continue until all the bones are excised.

  5. Place obtained bones with 5 ml HBSS containing 2% FBS in the mortar. Crush bones gently with pestle, using only enough force to crack open the bones. Agitate gently to release loosely adherent BM cells from bone chips and pipette off the supernatant. The supernatant containing BM cells can be filtered through a 100 μm cell strainer into a 50 ml conical tube. Add 10 ml fresh HBSS without FBS, and repeat agitation and removal of supernatant filtered through the 100 μm cell strainer into the same 50 ml conical tube. Repeat wash step with HBSS without FBS until the chips become pale, and try to filter all the washes into the same 50 ml tube. Leave the tube on ice for later use (see Notes 2 and 3).

  6. Transfer the bone chips to a 100 mm cell culture petri dish. Add 2 ml of collagenase II solution. Ensure all bone chips are completely covered in solution. Let them sit for 5 min. This step softens the bone, allowing it to be chopped more easily.

  7. Cut the remaining bone chips into small pieces of approximately 1–2 mm2 with a scalpel (see Note 4).

  8. Transfer the bone fragments and collagenase II solution to a new 50 ml conical tube, and add more collagenase II solution to a final volume of 20 ml per mouse.

  9. Seal lid and place the 50 ml tube containing bone chips from step 8 in a 37 °C shaking water bath at maximum speed for 1 h.

  10. Take the 50 ml tube containing the BM washes from step 5, and centrifuge at 400 × g, 4 °C for 10 min. Resuspend cells in 5 ml red cell lysis buffer, and incubate on ice for 10 min. Centrifuge cells at 400 × g, 4 °C for 5 min, and wash once with HBSS+2% FBS.

  11. After 1 h, remove the 50 ml tube from the water bath. Collect supernatant and filter through a 100 μm cell strainer into a new 50 ml tube. Wash bone chips by mixing with an additional 20 ml of HBSS with 2% FBS. Filter the wash through the 100 μm strainer into the same 50 ml tube (for a final volume of 40 ml).

3.2. Staining BM Cells and Endosteal Stromal Cells for Flow Cytometry Analysis

  1. Centrifuge the endosteal stromal cells collected from Subheading 3.1, step 11, at 400 × g for 10 min at 4 °C, and carefully remove the supernatant. Combine the endosteal stromal cells with the BM hematopoietic cells from Subheading 3.1, step 10. Centrifuge cells at 400 × g, 4 °C for 10 min (see Notes 5 and 6).

  2. Discard supernatant and resuspend the cells in 10 ml HBSS +2% FBS. Count the cell number using a hemocytometer.

  3. Dispense seven aliquots with each containing 0.5 × 106 cells into seven FACS. These aliquots are used for controls (Control 1–7). Control 1 is for unstaining negative control. Control 2 is for 7-AAD viability marker staining. Controls 3–7 are used for single-color fluorescence staining. These controls are used for fluorescence compensation and gate setting. Aliquot 5 × 106 to 1 × 107 cells as the main sample to stain osteoblasts, MSCs, and ECs.

  4. Prepare antibody cocktails in HBSS+ 2% FBS for the main sample with the following antibodies: APC-CD45, APC-Ter119, Pacific Blue-CD31, PE-CD51, and PE-Cy7-Sca-1. Use 100 μl of HBSS with 2% FBS containing 1 μl of each antibody per 1 × 106 cells.

  5. Resuspend main sample cells in the antibody mixture, and resuspend the single-color control samples (Controls 3–7) in 100 μl of HBSS with 2% FBS containing 1 μl of single antibody. Keep samples on ice for 30 min to allow the antibody binding.

  6. Wash cells twice with 1 ml cold HBSS +2% FBS per 1× 106 cells, and spin down at 400 × g for 5 min at 4 °C in between washes. Resuspend the main sample cells in HBSS+2% FBS at a density of 10 × 106/ml, add 5 μl 7-AAD per 100 μl HBSS+2% FBS, and filter through 100 μm cell strainer into 5 ml polystyrene round-bottom FACS tubes. Keep samples on ice. Resuspend cells in control tubes in 200 μl HBSS+2% FBS. Add 5 μl 7-AAD into Control 2. All samples are ready for FACS analysis.

  7. FACS BD™ LSR II is used for flow cytometry analysis. Dead cells are defined as 7AAD+ population. Osteoblasts are analyzed by gating on CD45-Ter119-CD31-CD51 + Sca1− population. MSC cells are selected by gating on CD45-Ter119- CD31 − CD51 + Sca1+. ECs are selected by gating on CD45-Ter119- CD31 + Sca1+ (Fig. 3).

Fig. 3.

Fig. 3

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Flow cytometry profiles and gate settings for osteoblasts, MSCs, and endothelial cells (ECs). (a) Mononucleated cells derived from endosteal niche and marrow compartment. (b) Selection of alive cells with 7-AAD staining (7-AAD negative cells). (c) Depletion of hematopoietic and red blood cells by negative selection of CD45-Ter119- (CD45 is pan-cell surface marker for nucleated hematopoietic cells; Ter119 is to label red blood cells). Non-hematopoietic cells (CD45-Ter119-) are further divided into two populations based on CD31 expression. CD31-negative cells are used for osteoblast (OB) and MSC staining, and CD31-positive cells are for further selection of ECs. (d) Identification of OBs and MSCs. The CD31- population (CD45-Ter119-CD31-) is selected for OBs and MSCs with positive expression of CD51 but negative and positive Sca-1 expression, respectively (CD45-Ter119-CD31 + CD51 + Sca-1- OBs and CD45-Ter119-CD31 + CD51 + Sca-1+ MSCs). (e) Identification of ECs. CD45-Ter119-CD31+ cells from panel C are selected for positive expression of Sca-1 to label ECs (CD45-Ter119-CD31 + Sca-1+)

3.3. Staining the Endosteal Stromal Cells for Flow Cytometry Sorting

  1. Centrifuge the endosteal stromal cells collected from Subheading 3.1, step 11, at 400 × g for 10 min at 4 °C, and carefully remove the supernatant. Resuspend the cells in 2 ml HBSS +2% FBS. Determine cell number. Count cells using a hemocytometer. The cells are ready for staining (see Notes 5 and 6).

  2. Dispense seven aliquots of 0.1 × 106 cells into seven FACS tubes that will be used for non-staining, 7-AAD viability marker staining, and single-color controls, respectively. The remaining cells are used as main sample to stain osteoblasts, MSCs, and ECs.

  3. Follow the Subheading 3.2, steps 4 and 6.

  4. FACS Aria™ II is used to sort the osteoblasts, MSCs, and ECs. The gate is set as Subheading 3.2, step 7.

3.4. HSCs and Stromal Cells Co-culture

  1. 2000 sorted osteoblasts (CD45-Ter119-CD31-CD51 + Sca1−) or MSCs (CD45 Ter119-CD31 − CD51 + Sca1+) are put into each well of 96-well plate. Cells are cultured in 200 μl co-culture medium at 37 °C in a 5% CO2 incubator and grown until confluence.

  2. Upon confluence, nonadherent cells are washed once with warm co-culture medium. Purified HSCs are sorted directly onto the adherent osteoblasts or MSCs and grown in co-culture medium supplemented with SCF (100 ng/ml), Flt3-L (100 ng/ml), and IL11 (25 ng/ml). Half amount of the culture medium is replaced after 2 days.

  3. After 4 days, all cells (including osteoblasts or MSCs) are harvested by vigorous pipetting to obtain the maximum number of cells. Hematopoietic cells are then stained with CD45 antibody, counted, and subject to functional analysis.

Acknowledgments

The authors are supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under awards R01HL124015 (YL), R21HL140213 (YL), and R43AI45726 and the Markey Cancer Center’s Flow Cytometry and Immune Monitoring Core Shared Resource Facility (P30CA177558). We thank the Markey Cancer Center’s Research Communications Office for editing and graphics support.

Footnotes

1.

Wet the whole body of the mouse with 70% ethanol thoroughly, and then put the mouse in sterilized hood and all the following steps are performed in the hood. To avoid microbial contamination of processed cells, especially cells that are subject to the long-term culture, do not allow bones to touch the mouse skin during dissection.

2.

Use minimal downward pressure to crack open the bones. Do not pulverize the bones, and try to use the least amount of force possible. If too much force is used, cell viability will decrease and excessive debris will be generated.

3.

Bone chips must be washed several times with HBSS without FBS before collagenase II solution is used to digest them. FBS reduces the activity of collagenase II, resulting in ineffective digestion and dissociation of stromal cells and reduced efficiency of cell procurement.

4.

Proper size of bone fragmentation is required to release adequate amounts of cells for cell separation.

5.

BM cells and endosteal stromal cells both contain MSCs [60]. Majority of MSCs are located in the arterial perivascular space near the inner surface of the cortical bone [61]. Less than 1% of BM cells contain MSCs. For analysis purpose, combine bone components that contain both endosteal cells and BM cells for more accurate frequency and numbers. However, for sorting purpose, use only endosteal cells to avoid contamination of BM hematopoietic cells and increase the purity of sorted cells.

6.

For flow cytometry analysis of osteoblasts, MSCs, and ECs, we perform all procedures in individual mouse. For stromal cell sorting, cells are pooled from several mice to obtain a sufficient number of cells; pooled cells are then subject to staining and flow cytometry sorting.

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