Use of an alpha-smooth muscle actin (SMAA) GFP reporter to identify an osteoprogenitor population

Osteoblast ablation model

The Col2.3Δtk mouse harbors a truncated version of the tk gene under the control of a 2.3 kb segment of the rat collagen type I promoter gene [16]. Utilizing this transgenic mouse we can selectively eliminate mature cells within osteoprogenitor lineage. To achieve this, 6–7 week old mice were injected intraperitoneally with 8 mg/kg/d of gancyclovir (Cytovene-IV; Roche Pharmaceutical, Nutley, NJ, USA) twice daily for 16-days. After the discontinuation of treatment, experimental mice were allowed to recover. During the entire experimental procedure, all mice were provided with crushed food and sacrificed at time point of 0, 4, 7, 12,17, 21 and 28 days of recovery.

Osteoblast lineage directed markers

To define cells as mature osteoblasts we utilized a transgenic mouse in which a 2.3 kb collagen type I promoter directs the expression of cyan or emerald variants of GFP to mature osteoblast lineage cells (Col2.3GFP(green) or Col2.3CFP(blue) [17, 18]. Col3.6GFPtpz directs expression to cells at the stage of a preosteoblast in vitro, and to periosteal fibroblasts. Following osteogenic induction, Col3.6GFP cells have the potential to differentiate into mature osteoblasts. We crossed homozygous Col2.3Δtk mice with either homozygous Col3.6GFP or homozygous Col2.3GFP mice. Animals heterozygous for both transgenes (tk and GFP) were used.

Paraffin histological methods

Femurs were fixed in 4% paraformaldehyde at +4°C for 7 days. Following fixation, they were decalcified in 15% ethylene-diamine tetraacetic acid (EDTA) for 3–4 days at room temperature, dehydrated with increasing concentrations of ethanol and cleared in xylene. Samples were embedded in paraffin (paraplast X-TRA tissue embedding medium, Fisher, Pittsburg) at 56°C and 5 µm thick sections were deparaffinized in xylene, hydrated in descending concentrations of ethanol and 2 mM MgCl₂ in PBS. Glycerol/PBS mounted slides were examined by fluorescent microscopy using dual FITC/Texas red filter cube of the Zeiss Axioplan 200 microscope [21]. The same sections were subsequently stained with hematoxylin and eosin.

μ-CT analysis

Analysis of bone geometry is performed at the University of Connecticut Health Center μ-CT facility. Mouse femur morphometry was assessed using conebeam micro-focus X-ray computed tomography (µCT40, Scanco Medical AG, Bassersdorf, Switzerland). Serial tomographic images were acquired at 55 kV and 145 µA, collecting 1000 projections per rotation at 300 msec integration time. Three-dimensional 16-bit grayscale images were reconstructed using standard convolution back-projection algorithms with Shepp and Logan filtering, and rendered within a 12.3 mm field of view at a discrete density of 578,704 voxels/mm³ (isometric 12-µm voxels). Segmentation of bone from marrow and soft tissue was performed in conjunction with a constrained Gaussian filter to reduce noise, applying a hydroxyapatite-equivalent density threshold of 470 mg/cm³. Three mice were used per time period.

Preparation of cryosections

Bones were fixed for 3–4 days in 4% paraformaldehyde/PBS (pH 7.4) at 4°C and decalcified 3 days as described above. Thereafter they were placed in 30% sucrose/PBS overnight and embedded in frozen specimen embedding media (Cryomatrix, ThermoShandon, Pittsburgh, USA) at −60°C. Adipose tissues were fixed overnight and placed in sucrose overnight and then embedded and cryosectioned. Bones and soft tissues were cryosectioned in 5µm sections using a CryoJane tape transfer system (Instrumedics, NJ, USA). After rehydration in 1 mM MgCl₂/physiological saline, GFP expression was observed and photographed using a Zeiss Axiovert 200M microscope and an Axiocam digital camera. A GFP-variant specific filters were utilized; GFP/Texas Red dual filter cube for visualization of green fluorescent protein, and Cyan/Texas red dual cube for blue fluorescent protein as decribed [21]. The filters were obtained from Chroma (Rockingham, VT). The dual bandpass design is required to distinguish the color of the GFP signal from the autofluorescence of the bone and bone marrow.

Immunostaining and Hematoxyillin Eosin (HE) staining

Following detection and imaging of GFP expression, slides were soaked in PBS for 30 min. to remove the coverslides. Sections were blocked with 1%BSA in PBS for 30 min. at room temperature. Sections were stained with Cy3-conjugated mouse monoclonal anti- α-smooth muscle actin (clone 1A, SIGMA, St. Louis, MO, USA) antibody for 1 hr at 1:800 dilution. We utilized monochorome filters to detect transgene driven GFP expression (Col3.6 and Col2.3GFP) in relation to α-SMA immunostaining (FITC filter, excitation 500/20, emission 535/30; TRITC filter, excitation 545/30, emission 620/60). Images were pseudocolored and composite images were generated using Photoshop. The number of single GFP expressing cells (α-SMA⁺ or Col3.6GFP⁺) or dual expressing cells were counted. Expression was evaluated in an area that is 2mm distal from growth plate and in proximity to the endocortical surface (selected area was 0.4mm²).

To detect endothelial cells we utilized immunohistochemistry for CD31 (PECAM). Briefly, sections were washed in PBS and then placed in 3% H₂O₂ for 30 min, followed by a blocking procedure at room temp for 20min. Sections were incubated with rat anti-mouse CD31 primary antibody at 1:100 dilution (BD Biosciences, San Jose, CA, USA) at 4°C overnight. Rabbit biotinylated anti-rat secondary antibody (Vector laboratories, Burlingame, CA, USA) was used at 1:200 dilution. Detection was achieved using a Vectastain ABC kit followed by DAB reaction according to manufacturer instructions (Vector laboratories). After immunostaining sections were washed, mounted with 50% glycerol and imaged. Then HE staining was done to indicate localization of GFP positive cells.

Bone marrow stromal cell cultures (BMSC)

Four-to 6-week old mice were sacrificed by CO₂ asphyxiation. Femurs and tibiae were dissected from surrounding tissues. The epiphyseal growth plates were removed and the marrow was collected by flushing with αMEM culture medium containing 100 U/ml Penicillin, 100 µg/ml streptomycin and 10% FCS, Hyclone (serum lot was previously tested for optimal osteogenic differentiation) with a 25 gauge needle. Single cell suspensions were prepared by passing the cell clumps through an 18-gauge needle followed by filtration through a 70-µm strainer and cells were plated at a density 1 × 10⁶ cells/cm².

Adipose derived stromal cell cultures (ADSC)

Adipose tissue was dissected from inguinal fat pads of 2–3 month old mice. Briefly, after removal, tissue was subjected to sequential 75 min digestions in an enzyme mixture containing Collagenase A (Roche) at a concentration of 0.15%. Cell digestion was resuspended with syringe and 18G needle, filtered using 75µm cell strainer, centrifuged, and resuspended in DMEM containing 10% FCS (serum lot was previously tested for optimal osteogenic differentiation). Cells were plated in 100mm dishes (8–10×10⁶/per plate) in DMEM containing 10% FCS.

Induction of osteogenesis and adipogenesis

Osteogenesis was induced by addition of 10⁻⁸M dexamethasone, 50µg/ml ascorbic acid and 8mM β-glycerol phosphate on day 7 of culture and every 2 days for a period of 2 weeks. Adipogenic induction was obtained by adding a cocktail of 0.5µM rosiglitazone and 1µM insulin for a period of 7 days.

Histochemical analysis of cell cultures

Histochemical staining for ALP activity was performed using a commercially available kit (86-R Alkaline Phosphatase, Sigma Diagnostics, Inc. ST. Louis, MO, U.S.A.) according to the manufacturer's instructions. Mineralization was assessed using a modified von Kossa silver nitrate staining method. Detection of lipid vacuoles was done using an Oil-Red-O (Sigma, USA). Stock solution was prepared as 0.5% in 100% isopropanol, and then fresh diluted to 0.3% before use. Cells were fixed in neutral buffer formalin, and stained for 1 hr. at room temperature. Images were acquired using a flat scanner and processed into a composite image using Adobe Photoshop.

Fluorescent microscopy of cultured cells

GFP expression in cell culture was visualized using an Olympus IX50 inverted system microscope equipped with IX-FLA inverted reflected light fluorescence (Olympus America Inc., Melville, NY). A specific excitation wavelength was obtained using FITC filter (exciter: 470/40; emitter: D525/50) to evaluate the expression of the α-SMAGFP. Cultures derived from dual α-SMAGFP/Col2.3GFPcyan mice were imaged using following filters: (GFP; excitation 500/20, emission 535/30) and (CFP; excitation 436/20, emission 480/40) to separate GFP from CFP. Images were recorded with a SPOT-camera (Diagnostic Instruments, Inc., Sterling Heights, MI).

Preparation of cell suspension

Freshly isolated adipose derived cells derived from α-SMAGFP mice were prepared as described in preparation of ADSC cultures. In addition, cells were washed one time in PBS and filtered into a new tube using a 75µm cell strainer to remove adipose tissue residues. Samples of ADSC derived from α-SMAGFP mice were obtained from 6-day-old culture by digesting the cells with 0.2% collagenase A/0.2% hyalouronidase for 20 min. Cells were centrifuged and washed in PBS. After washing, red blood cells were lysed using ammonium chloride.

Antibodies

The expression of α-SMAGFP was observed in context of the following combinations; a) α-SMAGFP⁺, anti-mouse CD45R-APC (clone RA3-6B2) and anti-mouse CD11b-PE (clone M1/70); b) α-SMAGFP, anti-mouse CD45R-APC, anti-mouse CD117-PE (clone 2B8); c) α-SMAGFP, anti-mouse CD45R-APC, anti mouse Sca1-PE or d) α-SMAGFP, anti-mouse CD45R-APC and anti mouse CD90-PE. All antibodies reagents were purchased from commercial sources (Pharmingen and eBioscience). Flow cytometry was done on a BD FacsClibur cytometer and data were processed using Cell Quest software.

Cell sorting

Offspring of dual transgenic mice were established by breeding the α-SMAGFP(green) with Col2.3GFPcyan (blue) to have the ability to identify populations of progenitor cells by expressing only SMAA-GFP and to follow their differentiation into osteoblast lineage by activation of Col2.3GFPcyan. ADSC from dual transgenic mice were cultured for 7 days to expand a population of progenitors and then were prepared for cell sorting to selectively purify α-SMAGFP⁺/Col2.3cyan⁻ populations. The population of interest was sorted by FACS using a BD Vantage SE with FACSDiva option (BD Biosciences). After sorting, purity of the isolated population was assessed.

Preparation of recipient mice

Wild type recipient mice received a lethal dose of 900 cGy total body gamma irradiation (TBI) using a 137Cs source (Nordion Gammacell 40 Irradiator; Atomic Energy of Canada, Ottawa, Canada), with a dose rate of 66.5 cGy/min. Rescue of the hematopoietic system of irradiated mice was performed by systemic infusion of 2.5×10⁶/100µl of donor total bone marrow cells derived from GFP negative (TBMc) into the retro-orbital sinus of anesthetized animals.

Transplantation procedure

Local injection into the medullary space of the tibia or femur was performed in ketamine/xylazine-anesthetized recipient mice. The knee was flexed to 90° and a 26-gauge needle was inserted into the joint surface of the femur or tibia through the patellar tendon and then inserted into the medullary space. The needle was removed and another 26-gauge needle attached to a 1-mL syringe was inserted into the needle tract and extended into the bone marrow space. Donor cells (α-SMAGFP⁺), 5×10⁵/20µL were slowly injected into the marrow cavity as the needle was being withdrawn from the intramedullary space [26]. Four wild type mice were used to evaluate the osteogenic potential of α-SMAGFP⁺ cells. To detect new bone formation in relation to the specific donor cell population, mice received a 100µL intraperitoneal injection of freshly prepared xylenol orange (XO; 30 mg/ ml, 90 mg/kg). Images were obtained using FITC/Texas Red and Cyan/Texas red dual cube as previously described [21].