A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Summary

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Abstract

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Introduction

Musculoskeletal diseases affect millions of people in the United States and present severe consequences for national and local health systems¹. These disorders are characterized by loss of bone and joint function that require extensive treatment and long periods of recovery. Commonly, a relative increase in the number and/or activity of osteoclasts, cells specialized to resorb bone, in osteoporosis and arthritis is observed². Under physiological conditions the number and activity of osteoclasts is regulated by receptor activator of nuclear factor κ-B ligand (RANKL), which is produced by osteoblasts. Osteoprotegerin (OPG), a decoy receptor for RANKL is also produced by osteoblasts³. In vivo animal models that involve systemic overexpression of sRANKL, or deletion of OPG are very valuable in osteoporosis research; however, these methods require the generation of transgenic mice^4,5. Here, a novel alternative method of overexpressing sRANKL for the study of musculoskeletal-related disorders is described. Specifically, minicircle (MC) DNA technology and hydrodynamic delivery methods were used to achieve gene transfer of sRANKL in vivo and overexpress mouse sRANKL systemically⁶.

This method is also complementary to other in vivo models of osteoporosis, such as hormonal modulation of osteoclasts following ovariectomy⁷ and dietary intervention by low-calcium diet⁸. These models are very useful to study different aspects of musculoskeletal-related disorders however they require surgical procedures and may take up to several months, at a significant cost⁹. Ovariectomized (OVX) rodent model is an experimental animal model where removal of ovaries leads to estrogen deficiency thereby mimicking human postmenopausal osteoporosis¹⁰. Human post-menopausal osteoporosis, a condition where estrogen deficiency leads to increased risk of bone fractures and osteoporosis affects approximately eight million women in the United States alone. Although the OVX model is useful for post-menopausal osteoporosis it offers limited advantages in studying osteoporosis in general. Estrogen suppresses bone loss, by inducing osteoclast and inhibiting osteoblast apoptosis, therefore in its absence an increased osteoclast activity is observed^10-12. A RANKL-OPG ratio imbalance that favors bone resorption is also observed¹³. However, estrogen deficiency in vivo is also accompanied by decreased levels of transforming growth factor β (TGF β), increased interleukin-7 (IL-7) and TNF, IL-1 and IL-6^14,15. As these cytokines have known bone remodeling modulatory functions independent of the RANKL pathway, it is impossible to attribute any osteoclast activation solely to the RANKL-RANK axis. The model described in this paper enables researchers to study in vivo RANKL-RANK axis in osteoclastogenesis and bone loss without pro-inflammatory cytokines compared to OVX rodent models.

Additionally, in vitro osteoclastogenesis techniques are essential tools to study osteoclast activation for potential therapeutic treatments of musculoskeletal diseases. Previous studies have also shown that culturing mouse bone marrow derived macrophages (BMMs) with mouse macrophage colony-stimulating factor (M-CSF) and mouse sRANKL can lead to osteoclast differentiation^3,16,17. Here, the protocols to generate multinucleated osteoclast-like cells from mouse bone marrow as well as from human peripheral blood mononuclear cells (PBMCs) in vitro¹⁸ are described. The cell-based assays required to define a mature terminally differentiated and fully functional osteoclast are also briefly described. These in vitro techniques complement the novel in vivo approach and together serve as powerful investigative tools to study osteoclast differentiation and activation. Using these systems, scientists are able to generate osteoclasts in vivo and in vitro and define the stimuli and signals required for their proliferation and activation as well as test the efficacy of pharmacological and biological inhibitors.

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Protocol

1. Hydrodynamic Delivery of sRANKL MC DNA

1. Hydrodynamic Delivery via Mouse Tail Vein
   1. Weigh the mouse before the tail vein injection. Dilute sRANKL or green fluorescent protein (GFP) MC in Ringer's solution (pre-warm at 37 °C) in a total volume of ~10% of the mouse's body weight.
   2. Warm up the mouse in a cage for 10 min prior to injection in order to dilate blood vessels and make lateral veins (LVs) visible. Monitor the mouse carefully to avoid dehydration and hyperthermia.
   3. As soon as the LVs are dilated and visible, transfer the mouse to the restrainer and insert the plug far enough into the barrel to restrain movement. Monitor the mouse for normal activity such as breathing. Adjust the plug of restrainer if needed.
   4. Disinfect the injection area 3/4 from the tip of the tail with an alcohol wipe. Use a 27-30 G needle for mouse tail vein injection. Hold the tail firmly with one hand and insert the needle into the tail vein. Apply pressure on the syringe and complete the injection within 5-7 sec.
   5. Remove the needle from the tail vein and apply pressure with a cotton ball on the injection site to stop bleeding. Monitor the mouse for 15-30 min, then transfer the mouse back to vivarium once the breathing rate is reduced to its normal rate.
2. Confirmation of Systemic Mouse sRANKL Expression and Quantification of Mouse Serum TRACP5b Post Gene Transfer
   1. Warm up the mouse in a cage for 5-10 min in order to dilate blood vessels and make LVs visible. Monitor the mouse carefully to avoid dehydration and hyperthermia.
   2. Make a small incision with a blade to the mouse's tail at a site one forth from the tip of the tail.
   3. Collect approximately 100 μl of blood in serum separation tubes.
   4. Incubate serum separation tubes at room temperature for 30 min.
   5. Centrifuge samples at 10,000 x g for 5 min and collect serum. Store serum in -80 °C for further analysis.
   6. Perform sRANKL ELISA and mouse serum TRACP5b assay on serum samples using a commercial available kit.

2. In vitro Osteoclast Generation from Mouse BMMs

1. Isolation and Culturing of BMMs
   1. Isolation of the tibia and femur bones: Euthanize the donor mouse with CO₂, and disinfect the legs with 70% ethanol. Dissect from the pubic bone to the calcaneus bone, to remove the femur and tibia bones intact.
   2. Remove the skin and muscle carefully without damaging the bone. Place processed femur and tibia bone in a Petri dish containing phosphate buffered saline (PBS).
   3. Removal of bone marrow: Cut off the tip on one side of the femur and tibia bones and flush out the bone marrow into a 50 ml tube using a 1ml syringe with a 25 G needle, loaded with α-MEM containing 1% penicillin/streptomycin (Osteoclast Culture Medium/OCM).
   4. Process bone marrow: Mix and transfer bone marrow suspension to a new 50 ml tube by passing it through a 70 μm nylon cell strainer. Centrifuge the cells at 300 x g and re-suspend in α-MEM containing 1% penicillin/streptomycin and 10% FBS (Osteoclast Culture medium/OCM (+)).
2. Culture of Macrophages
   1. Count cells using a hemocytometer or automatic cell counter.
   2. Plate 1 x 10⁶ cells per well for a 6-well plate or 3 x 10⁵ cells to glass coverslips (5 mm) or dentine slices placed in 96-well plate and incubate at 37 °C.
   3. Aspirate all media containing non-adherent cells and incubate cells at 37 °C with fresh OCM (+) containing 25 ng/ml mouse M-CSF for 48 hr.
   4. Aspirate all media and incubate cells at 37 °C with fresh OCM (+) media containing 25 ng/ml mouse M-CSF, and 30 ng/ml mouse sRANKL to initiate osteoclastogenesis. Replenish media every 3 days as required.
   5. Grow cells for approximately 6-8 days to form giant multi-nuclear cells capable of resorbing bone. As soon as multinucleated cells appear, perform TRAP (tartrate resistant acid phosphatase) staining using a commercial available kit, and when fully matured, perform functional assays. Visualize F-actin rings on coverslips using phalloidin stain and image the cells attached to dentine slices by scanning electron microscopy (SEM) as described previously¹⁹.

3. In vitro Osteoclast Generation from Human PBMCs

1. Isolation of Human PBMCs
   1. Add 10 ml of pre-warmed Histopaque-1077 into a 50 ml tube.
   2. Empty leukocyte filter obtained from blood bank with sterile PBS into a 50 ml tube (1:1 ratio of blood with PBS). Layer the diluted blood over the histopaque solution very slowly, making sure that the blood does not mix with the histopaque.
   3. Centrifuge samples at 1000 x g for 20 min at 18 °C. Following centrifugation, carefully isolate the white buffy coat layer containing the white blood cells and transfer to a new 50 ml tube.
   4. Dilute cells with PBS and centrifuge again at 650 x g for 5 min to collect the cells.
   5. Remove supernatant and resuspend cells in OCM (-) media and count cells using cell counter.
2. Plating and Culturing PBMCs
   1. Resuspend the cells in OCM (+) media and plate 8 x 10⁶ cells/ml per well in a 6-well plate or 1 x 10⁶ cells/ml per well in 96-well plate. Plate cells on glass coverslips (5 mm) as required for immunofluorescence imaging as well as on bone slices suitable for bone resorption assays as previously described²⁰.
   2. Change culture media, replenishing the cells with human M-CSF every 2-3 days or as required. Then proceed to differentiation step on day 5 by adding 30 ng/ml of human sRANKL along with 25 ng/ml human M-CSF in OCM (+) media to initiate osteoclastogenesis.
   3. Grow cells for approximately 14 to 21 days to form giant multi-nuclear cells. As soon as multinucleated cells appear, these cells can then be labeled for TRAP, and when fully matured, functional assays can be performed. F-actin rings can be visualized on coverslips using phalloidin stain. The cell morphology can be examined in cells attached to dentine slices by SEM.

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Results

Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. In Figure 1, the representative results of successful gene transfer of GFP and mouse sRANKL MC in mice are shown. In Figure 2, the representative images of mouse bone marrow or human PBMC cell differentiation time course ...

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Discussion

Musculoskeletal conditions are leading causes of morbidity and disability and are comprised of over 150 diseases and syndromes; affecting approximately 90 million Americans today. Joint inflammation and bone destruction are predominant features of musculoskeletal conditions, including arthritis and osteoporosis. Osteoporosis is a condition that weakens bone integrity, often leading to fractures of the bone. Arthritis is a chronic, debilitating disease characterized by inflammation of the joints that become swollen, tende...

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Materials

Name	Company	Catalog Number	Comments
alpha-MEM	Life Technologies	12561-056
Human M-CSF	Miltenyi Biotec	130-096-492
Mouse M-CSF	Miltenyi Biotec	130-094-643
Human RANK-Ligand - soluble	Miltenyi Biotec	130-094-631
Mouse RANK-Ligand - soluble	Miltenyi Biotec	130-094-076
Tailveiner Restrainer for mice	Braintree	TV-150 STD
Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit	R&D systems	MTR00
Acid Phosphatase, Leukocyte (TRAP) Kit	Sigma	387A
MouseTRAP assay	immunodiagnostic systems	SB-TR103