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[Abstract] [Full Text] [PDF]
An in vitro method to study the effects of hematopoietic regulators during immune and blood cell development Nitixa Patel1, Marianne Castillo1 and Pranela Rameshwar1* 1 Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ. UMDNJ-New Jersey Medical School, MSB, Rm. E-579 185 South Orange Ave, Newark, NJ 07103, USA. New Jersey.
Phone: +(973) 972 0625
Fax: +(973) 972 8854
Email: rameshwa@umdnj.edu.
Biol. Proced. Online 2007;9:56-64. doi:10.1251/bpo133 Indexing terms: cell culture techniques; hematopoiesis; cytokines; hematopoietic stem cells. Abbreviations: BM, Bone marrow; CLP, common lymphoid progenitors; CMP, common myeloid progenitors; HSCs, hematopoietic stem cells; MSCs, mesenchymal stem cells; NK-A, neurokinin-A; NK1, neurokinin-1; NK2, neurokinin-2; SDF-1α, Stromal cell-derived factor 1α; SP, Substance P. |
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34+ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.
Hematopoiesis is the process by which hematopoietic stem cells (HSCs) self-renew to form blood and immune cells of the body. In the healthy adult, the major site of hematopoiesis is the bone marrow (BM) (1,2). HSCs differentiate into common myeloid progenitor (CMP) or common lymphoid progenitor (CLP). CMLs differentiate into multiple lineages to produce erythrocytes, monocytes, megakaryocytes, dendritic cells and granulocytes (3). CLPs generate lymphoid cells, T- and B cells, natural killer cells and dendritic cells (4,5). In contrast, dysfunctional BM shows extra-medullary hematopoiesis, such as in spleen (6). HSCs are mainly found in areas closest to the endosteum of BM in the region of lowest oxygen level (7). The BM is also host to another stem cell, mesenchymal stem cells (MSCs), which is located surrounding blood vessels and in contact with trabeculae (8). MSCs are functionally linked to HSCs since the supporting stromal cells for the latter are generated from MSCs (9,10). Hematopoiesis is regulated by a complex system involving soluble factors such as cytokines, chemokines, neuropeptides and neurotransmitters (11). Extracellular matrix proteins (ECM) are also involved in hematopoietic regulation and are not mutually exclusive of cytokines (11,12). There is no direct link between families of regulator with functions since the BM microenvironment could influence hematopoietic outcome (11,12). Thus, hematopoietic regulators involve a complex network involving cellular interactions in a network of soluble factors and ECM. This study focuses on a method to study two regulators of hematopoiesis, stromal-derived growth factor (SDF)-1α and the preprotachykinin I gene, also referred to as Tac1. SDF-1α belongs to the CXC chemokines family, and interacts with the CXCR-4 receptor that belongs to the family of G-protein, seven transmembrane receptors (13). SDF-1α is produced by BM stromal cells and could be released or membrane-bound (14,15). CXCR4 receptors are expressed on HSCs, making them poised to form complexes with stromal cells through interactions with membrane-bound SDF-1α (14,15). Disruption of the SDF-1α-CXCR4 complex could cause mobilization of HSCs from BM into the periphery (14). SDF-1α levels show a pattern of gradient changes across the BM (16). Gradient changes in SDF-1α across the BM facilitate mobilization of HSCs in and out of the BM (14). In addition to its mobilization role, SDF-1α has a major role in hematopoiesis at all levels of maturity within the hematopoietic lineages (17,18). Tac1 encodes peptides belonging to the tachykinin family of peptides (19). Tachykinins presence in BM could be from the innervation in BM as neurotransmitters and from endogenous sources (20,21). The major Tac1 peptides are substance P (SP) and neurokinin-A (NK-A) (22). Substance P induces the production of cytokines with stimulatory effects on hematopoiesis whereas neurokinin-A acts in opposing manner (Table 1). SP and NK-A bind the neurokinin (NK) receptors with varying affinities (23). Three NK receptors have been described, NK1-NK3 (24). This family of receptors belongs to the 7-transmembrane G-protein coupled receptor family (25). SP shows preference for NK1 and NKA demonstrates high affinity binding to NK2 (23). NK receptors are expressed on hematopoietic progenitors and differentiated immune cells (20). The hematopoietic effects mediated by NK1 and NK2 are confounded by crosstalk between these receptors, and fragments of Tac1 peptides that bind NK1 (22). We have reported Tac1 expression by SDF-1α in the BM stroma (19). This production was important in SP-mediated effects on hematopoiesis (19). The hematopoietic effects were examined as the levels of primitive and mature hematopoietic progenitors by long-term culture-initiating cell assay and clonogenic assay (19). Here we describe detailed methods to investigate hematopoiesis through secondary regulators and the incorporation of stromal cells.
Reagents and cytokinesα-Minimum Essential Medium (α-MEM), glutamine, hydrocortisone, SP, and phthalic acid dially ester were purchased from Sigma (St. Louis, MO). Fetal calf sera (FCS) and horse sera (HS) were purchased from Hyclone Laboratories (Logan, UT). Recombinant human SDF-1α was purchased from R&D Systems, prolyl-4-hydroxylase mAb from Dako Cytomation, PE-anti-CD14 from BD Pharmingen. Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was provided by the Immunology Department of Genetics Institute (Cambridge, MA). 125I-Tyr8-SP (2200 Ci/mmol/L) was purchased from Perkin Elmer (Billerica, MA). Dimethyl phthalate was purchased from Fisher Scientific, Pittsburgh, PA. Dynabead M-450 CD34 was purchased from DynaI Inc. (Lake Success, NY). PE-anti-CD34 was purchased from Becton Dickinson, San Jose, CA and Tri-color-anti-CD45 from Caltag Laboratories (Burlingame, CA). Primary bone marrow cellsBM aspirates were obtained from the posterior iliac crest of healthy volunteers following the guidelines made by the Institutional Review Board of the University of Medicine and Dentistry of New Jersey (Newark, NJ). BM aspirates were obtained from healthy donors between the ages of 18-25 years. Clonogenic assays for granulocyte-macrophage colony-forming units (CFU-GM)Mononuclear cells (BMNCs) were isolated from BM aspirates by Ficoll-Hypaque density gradient and then assayed for CFU-GMs in sera free culture as described (26). 105 mononuclear cells/ml were plated in methylcellulose with different concentrations of Substance P (SP) and 3 U/mL rhGM-CSF. Colonies with more >20 cells were counted at day 10. Preparation of BM Stroma107 nucleated cells from BM Aspirates were added to 25 cm2 tissue culture flasks (Falcon 3109) in stromal media (α-MEM with 20% FCS) and incubated at 370 C for 3 days. Mononuclear cells were then separated by Ficoll-Hypaque density gradient and replated in fresh stromal medium. Cells were incubated until confluency with weekly replacement of 50% stromal medium. At confluency, the trypsin-sensitive adherent cells were passaged at least 5 times before being used for experiments. Flow Cytometry studies indicated stromal cells were negative for CD14 and positive for prolyl 4-hydroxylase. Modified long-term culture-initiating cell (LTC-IC) cultureLTC-IC assays were performed as described (19). Stromal cells were cultured in 25-cm2 flasks and at confluence, were γ-irradiated with 150 Gy delivered by a cesium source. After 16 h, media were replaced with 5 ml of fresh media containing 107 BMNCs/flask. At weekly intervals, 50% of culture media were replaced. The non-adherent cells were studied at various intervals in clonogenic assays for CFU-GM, described above. Isolation of CD34+ BM cellsCD34+ cells were positively selected from BMNC with an isolation kit (Dynabeads M-450 CD34) purchased from DynaI Inc. (Lake Success, NY) with a 2-step method as described. BMNC (107-108) were washed twice and resuspended in 1 ml of cold isolation buffer (Ca2+/Mg2+ - free PBS with 2% BSA). 108 Dynabeads M-450 CD34 were incubated with the cell suspension at 4°C for 30 min using gentle agitation at 5 min-intervals. Dynabeads were then magnetically selected with Dyna1 MPC and then washed (3x) with isolation buffer. Cells were uncoupled from the magnetic beads by incubating at 37°C for 15 min with equal volumes of isolation buffer and DETACHaBEAD CD34 liquid. Cells were double labeled with PE-conjugated anti-CD34 and Tri-color-conjugated anti-CD45. The process was repeated and the final population was >90% positive for CD34 and CD45. Since endothelial cells also express CD34, the inclusion of this cell subset in the selected cell population was determined using anti-von Willebrand Factor. Less than 1% of selected cells were positive for vWF. Binding AssayVarying concentrations (3-200 mmol/L) of 125I-Tyr8-SP (2200 Ci/mmol/L) were added to 1ml of CD34+ cells and resuspended at 106/mL. Tubes were rotated at 4°C with a Rotating Wheel Two-way Mixer (Robbins Scientific, Sunnyvale, CA) for 4 hours in order to allow for binding. After this period of incubation, cells were layered in siliconized microcentrifuge tubes on 0.5 mL phthalate oils (90 parts dimethyl phthalate and 10 parts phthalic acid dially ester) and then centrifuged at 10,000 g for 1 min at 4°C. The cells were incubated for 4 h, which falls within the range required for equilibrium that was established in time course binding assays ranging from 1 to 12 h. The radioactivity was measured in the pellet and supernatant fractions in order to determine the amount of bound and unbound ligand, respectively. Radioactivity was determined using the Beckman gamma counter (Beckman Instruments, Irvine, CA). Triplicate binding were performed for each donor and concentration of ligand. The McPherson's modification of the Radioligand Binding Analysis software was used to analyze the data.
The role of Tac1 peptides as mediators within the neural-immune-hematopoietic axis has been widely studied (20,21).The hematopoietic effects of SP and NKA are mostly mediated through their interaction with NK1 and NK2, respectively (27,28). However, the effects of these peptides are not only direct, but indirectly through the production of cytokines and other hematopoietic regulations in BM resident cells (Table 1).
Experimentally, studies can be done to show effects of Tac1 peptides on hematopoiesis, at an early stage by LTC-IC assays (27-33). In addition to the early phase, SP and NKA also affect hematopoietic progenitors at the myeloid level, which was studied by clonogenic assays for CFU-GM (Table 1). The effects of tachykinins are not only mediated through cytokine production, but could also occur directly. This premise is based on the ability of SP to bind CD34+ BM cells at dissociation constant of 70 nM/L on a cell subset that includes both early and late human progenitors (Fig. 1).The methods described in this technique paper could be applied in studies to investigate the roles of cytokines in the network developed by Tac1 in BM stromal cells.
Fig. 2 summarizes how the methods could be applied to study the effects of SDF-1α on Tac1 expression, and the consequence to hematopoiesis. SDF-1α is expressed as baseline in stromal cells. Upon stimulation by exogenous SDF-1α, which could be produced by other accessory cells, and also by stroma, Tac1 expression is upregulated. This could lead to the production of SP, which could mediate direct stimulation on HSCs (Fig. 1), or indirectly by inducing the production of other cytokines in cells within BM microenvironment. These cytokines could stimulate hematopoiesis. To address questions similar to the network described in Fig. 2, experimental studies could be developed by clonogenic assays, LTC-IC assays and cytokine quantitation and mechanistic studies on cause-effect relationship could be done by knockdown and expression approaches (19).The effects of SP on the stimulation of primitive HSCs and progenitors have been attributed to the induction of specific hematopoietic growth factors including IL-3, IL-6, GM-CSF, SCF, flt3/flk2 (29). Similarly, NK-A, induces the production of specific cytokines with hematopoietic suppressive effects (30). We have reported the production of TGF-β and MIP-1α by NK-A in stromal cells (30). Together, the positive and negative effects of the tachykinins lead to an understanding of hematopoietic homeostasis.
Additionally, SDF-1α is shown to be involved in hematopoiesis (19). In BM, SDF-1α follows a gradient pattern with the highest levels close to the stromal compartment (32). CXCR4, expressed on HSCs mediates retention in BM (32). Changes in SDF-1α in BM could cause increased hematopoietic activity and also mobilization of HSCs. The molecules involved in this process could be identified using the methods described as the basis for questions.
This work was supported by grant awarded by the Department of Defense and UH-New Jersey Medical School Cancer Center.
Perform all procedures under sterile conditions. Step 1:A. Stromal Isolation, 2-step procedure:
Step 2:
Immunofluorescence Stain:
B. Clonogenic Assay:
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