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Murine embryonic stem (mES) cells are self-renewing pluripotent cells that bear the capacity to differentiate into ectoderm-, endoderm-, and mesoderm-derived tissues. In suspension culture, embryonic stem (ES) cells grow into spherical embryoid bodies (EBs) and are useful for the study of specific gene products in the development and function of various tissue types. Osteoclasts are hematopoietic stem cell-derived cells that participate in bone turnover by secreting resorptive molecules such as hydrochloric acid and acidic proteases, which degrade the bone extracellular matrix. Aberrant osteoclast function leads to dysplastic, erosive, and sclerosing bone diseases. Previous studies have reported the derivation of osteoclasts from mES cells; however, most of these protocols require coculture with stromal cell lines. We describe two simplified, novel methods of stromal cell-independent ES cell-derived osteoclast development.
Osteoclasts are cells of hematopoietic origin, specifically the monocyte–macrophage lineage, that play a critical role in bone remodeling and calcium and phosphorous homeostasis within the body . Osteoclastogenesis is supported by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), both of which are produced by mesenchymal cells in the bone marrow environment . Along with bone-building osteoblasts, they participate in bone turnover by secreting acid onto the bone surface [3, 4]. Osteoclasts are large, multinucleated syncytia that form from the fusing of mononuclear precursors and strongly express the enzyme tartrate-resistant acid phosphatase (TRAP).
Increased function of osteoclasts leads to dysplastic and erosive bone diseases including osteoporosis, Paget's disease of bone, bone metastases, and wear particle-induced osteolysis following arthroplasty [5–7]. Reduced osteoclast function can contribute to osteopetrosis and sclerosing bone diseases . Previous research has examined the differentiation of osteoclasts at different stages in their development with the hope that the results will provide insight into the pathophysiology of these conditions. Murine embryonic stem (mES) cells have emerged as a powerful tool to evaluate the development of multiple cell lines, including osteoclasts, as multiple genetic models have been created in the process of generating knock-out and knock-in mice. For mES cell-derived osteoclast development, previous published procedures have consisted largely of two- or three-step cocultures with the stromal cell lines OP9 or ST2 as feeder layers [9–14] with multiple growth factors and hormones in various combinations, including: dexamethasone, 1α, 25-dihydroxyvitamin D3, ascorbic acid, RANKL, M-CSF, and vascular endothelial growth factor (VEGF). Previous reports describing embryonic stem (ES) cell-derived osteoclast development in a stromal cell-free environment are limited and demonstrated a very low efficiency of osteoclast differentiation [11, 13]. We have developed two straight-forward, stromal cell-free methods of ES cell-derived osteoclast assays that can be effectively employed to study osteoclast development and differentiation.
mES cells were cultured at 37°C in 5% CO2 on gelatin-coated tissue culture plates in high-glucose (4.5 g/L) Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA), 2 mM l-glutamine (StemCell Technologies, Vancouver, BC, Canada), 100 U/mL/100 μg/mL penicillin/streptomycin (StemCell Technologies), 0.1 mM nonessential amino acids (StemCell Technologies), 1 mM sodium pyruvate (StemCell Technologies), 55 μM β-mercaptoethanol (Invitrogen), 15% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and 1000 U/mL leukemia inhibitory factor (LIF; ESGRO; CHEMICON, Temecula, CA, USA).
Undifferentiated ES cells were trypsinized, quantitated, and plated at 2,000 cells/mL in 0.9% methylcellulose-based media containing Iscove's Modified Dulbecco's Medium (IMDM, Invitrogen), 2 mM l-glutamine (StemCell Technologies), 100 U/mL/100 μg/mL penicillin/streptomycin (StemCell Technologies), 5% protein-free hybridoma medium (PFHM-II, Invitrogen), 50 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 200 μg/mL iron saturated holo-transferrin (Sigma-Aldrich, St. Louis, MO, USA), 450 μM monothioglycerol (Sigma-Aldrich), and 15% ES cell hematopoietic differentiation FBS (StemCell Technologies) in petri dishes. Cells were cultured for growth into embryoid bodies (EBs) at 37°C in 5% CO2 for 6 days (Fig. 1A).
EBs were collected and washed two times with PBS and digested in 0.25% trypsin-EDTA (Invitrogen) for 5 min at 37°C. Trypsin was quenched by the addition of serum and EBs were disaggregated by passage through a 20-gauge needle two times. Single cells were plated in osteoclast differentiation media including α-minimal essential medium (α-MEM, Invitrogen), 10% ES cell hematopoietic differentiation FBS (StemCell Technologies), 100 U/mL/100 μg/mL penicillin/streptomycin (StemCell Technologies), 30 ng/mL M-CSF (Peprotech, Rocky Hill, NJ, USA), and 50 ng/mL murine RANKL (M-RANKL, Peprotech) and plated at 250,000 cells/3.5 cm tissue culture plate in 3 mL of media. The cells were incubated for 7 days at 37°C in 5% CO2 with the media being changed every other day.
Trypsinized ES cells were resuspended at a concentration of 10,000 cells/mL in IMDM (Invitrogen), 2 mM l-glutamine (StemCell Technologies), 100 U/mL/100 μg/mL penicillin/streptomycin (StemCell Technologies), 5% PFHM-II (Invitrogen), 50 μg/mL ascorbic acid (Sigma-Aldrich), 200 μg/mL iron saturated holo-transferrin (Sigma-Aldrich), 450 μM monothioglycerol (Sigma-Aldrich), and 15% ES cell hematopoietic differentiation FBS (StemCell Technologies). To prepare hanging drops, 30 μL/drop of cell suspension (300 cells) was pipetted onto the inner portion of a tissue culture plate lid and the lid was gently placed on a tissue culture plate containing 5–10 mL of PBS to prevent desiccation of the drops (Fig. 1B). Hanging drops were cultured for 2 days at 37°C in 5% CO2.
Following 2 days, the EBs were collected by flooding the inner portion of the tissue culture lid with media and undissociated EBs were plated at approximately 40 EBs/6 mL in osteoclast differentiation media in 6 cm tissue culture plates. Under these conditions, preformed EBs adhered to the tissue culture plate and continued to undergo differentiation. Following 4 days in culture, cells were trypsinized, quantitated, and plated at 250,000 cells/3.5 cm tissue culture plates in 3 mL of osteoclast differentiation media and incubated for 7 days at 37°C in 5% CO2 with the media being changed every other day.
To stain for osteoclasts, cells were fixed with 10% formaldehyde in PBS for 10 min, fixed with a 50:50 ethanol–acetone solution for 1 min, and stained for TRAP. In brief, 5 mg of naphthol AS-MX phosphate (Sigma) was diluted in 0.5 mL of N,N-dimethylformamide (Wako, Richmond, VA, USA) and this solution was combined with 30 mg of fast red violet LB salt (Sigma) and 50 mL of 0.1 sodium acetate buffer (pH 5.0) containing 50 mM sodium tartrate . Cells that were TRAP-positive (purple) and contained 3+ nuclei were considered osteoclasts.
Both the methylcellulose-based and hanging drop differentiation successfully produced mature osteoclasts (Table 1 and Fig. 2). Regarding the methylcellulose-based differentiation (n=5), an average of 2204 TRAP+ cells per 250,000 cells plated were observed. Of the TRAP+ cells, an average of 381 was mature osteoclasts with ≥3 nuclei, accounting for 16% of all TRAP+ cells. Using hanging drop differentiation (n=3), greater numbers of TRAP+ cells per 250,000 cells plated were observed compared to the methylcellulose differentiation method (Table 1 and Fig. 2A). However, a similar percentage of TRAP+ cells fully differentiated into mature osteoclasts with ≥3 nuclei (17%, Table 1).
Previous studies have demonstrated the successful differentiation of mature osteoclasts from ES cells [9–13]. One method utilizing an induction culture, expansion culture, and osteoclast culture (total culture time 16 days) produced osteoclasts at a rate of ~1,375 osteoclasts/2,000 cells plated (69%); however, this high efficiency method required coculture with stromal cell lines (OP9 or ST2 cells) . A single step, stromal cell-free method of ES cell-derived osteoclast differentiation has been reported; however, the efficiency of TRAP+ cell generation was low, ranging from 0.025% to 0.1% of plated ES cells [11, 13]. To improve the efficiency of ES cell-derived osteoclast differentiation in a stromal cell-free culture, we utilized two methods of ES cell differentiation: methylcellulose-based and hanging drop differentiation, prior to plating cells in osteoclast differentiation media. Using these methods, we found that we could increase the efficiency of ES cell TRAP+ cell generation to 1–2% for methylcellulose-based and hanging drop differentiation, respectively. Thus, these methods of ES cell differentiation, commonly employed for hematopoietic progenitor and cardiomyocyte functional evaluation, can also be used for osteoclast developmental analysis. Our hope is that these methods will facilitate the understanding of the pathogenesis of osteoclast-related diseases.
This work was supported by the Department of Pediatrics, Indiana University School of Medicine and by the National Institutes of Health (RO1 HL082981, R.J.C.). The authors gratefully acknowledge the administrative assistance of Linda S. Henson.
Michael L. Goodman, Shi Chen, Feng-Chun Yang and Rebecca J. Chan: No competing financial interests exist.