Candidate antibodies were tested for reactivity and specificity with equine cell surface antigens. Subsequently, cell surface molecules of uncultured bone marrow cells were analyzed using flow cytometry. Bone marrow cells were cultured and harvested on 2, 7, 14, 21, and 30 days for analysis of cell surface proteins and gene expression. All procedures were performed in compliance with institutional guidelines for research on animals.
To validate reactivity of antibodies with equine cells, peripheral blood cells were used as positive and negative controls. Whole blood (30 mL) was collected from five horses for antibody validation. Blood samples were drawn into preservative free heparin to a final concentration of 33 units/mL. Candidate equine and human monoclonal antibodies tested are listed in . Whole venous blood was processed prior to flow cytometry analysis using density gradient centrifugation to remove the majority of red blood cells as previously described [12
Candidate Antibodies Tested to Determine the Changes in Equine Mesenchymal Progenitor Cells Surface Antigens in Uncultured Samples and Subsequent Propagation of Cells in Culture
Validation of antibody specificity
The CD44 and CD11a/CD18 antibodies have been previously validated as specific for their respective molecules in the horse [13
]. The CD11a/CD18 antibody (clone CZ3.2) identifies a noncovalently linked heterodimer consisting of a 180 kDa α-chain (CD11a) and a 95 kDa β-chain (CD18) using immunoprecipitation under reducing conditions [13
]. The CD44 antibody (clone CVS 18) identifies a heavily glycosylated molecule of 65–100 kDa [13
]. On a 12% SDS-PAGE analysis, a “smear” was produced approximately in the 100 kDa position, indicating that the precipitated molecule was heavily glycosylated. The analysis was repeated after endoglycosidase F treatment of the precipitate, and a single 76 kDa band was produced in both reducing and nonreducing conditions [13
]. The CD44 antibody has also been shown to react with protein produced by a cDNA-encoding equine CD44 molecule in a COS cell expression system [15
For CD90, CD29, and CD45RB antibody validation analyses, whole cell lysates were prepared from fresh peripheral blood leukocytes and from red blood cells with platelets. Western blot analyses were performed to determine if the reactive candidate antibodies bound proteins of the expected size based on previous literature, protein size similarity to other species, or predicted equine sequences. The CD90 antibody was expected to detect a ~17 kDa protein, similar in size to the equivalent human protein. Similarly, the CD29 antibody was expected to detect an ~130 kDa protein based on the size of the human protein. The CD45RB antibody was expected to have one or more bands <150 kDa based on the multiple isotypes of the human protein. To test the CD45RB and CD29 antibodies, proteins from cell lysates were resolved on 7.5% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels, which were subsequently transferred to polyvinylidene fluoride (PVDF) membranes and probed with the relevant antibody. A 15% SDS-PAGE gel was used to resolve cell lysates for subsequent analysis of the CD90 antibody following protein transfer to a PVDF membrane.
An immunoprecipitation was performed in addition to western blot analysis for the CD29 antibody, using an unconjugated version of the antihuman CD29 (Beckman Coulter, clone 4B4LDC9LDH8) used in this study. A 7.5% SDS-PAGE gel was used to resolve the immunoprecipitated products. Following protein transfer, the PVDF membrane was probed with antibody known to recognize human β1-integrin (Calbiochem, clone 4B7-CP26).
Bone marrow aspirate collection and cell isolation
Bone marrow aspirate was used to assess changes in cell surface markers over time and for tri-lineage (cartilage, bone, and adipose) differentiation. Bone marrow aspirate was collected from the sternabrae of 35 horses (11 males and 24 females, age range 6 months–20 years) under standing sedation with xylazine hydrochloride (0.55 mg/kg IV) and local anesthesia using 2% lidocaine hydrochloride (10 mL/site). Samples were collected in preservative-free heparin (American Pharmaceutical Partners Inc, Schaumburg, IL) to a final concentration of 33 units/mL.
Aspirate (60 mL) from each horse was diluted to 180 mL total volume using phosphate-buffered saline + 0.5% bovine serum albumin. The white blood cell fraction of the sample was enriched and the majority of red blood cells were removed by layering each 30 mL aliquot of dilute sample on Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ) for density gradient centrifugation, as described for antibody validation. Samples were resuspended in 50 mL MPC culture media (see below) prior to cell counting using a hemocytometer. Approximately 2–9 × 108 bone marrow mononuclear cells (BMMNC) were obtained per sample using this method. A portion (~10 × 106 cells) of the uncultured bone marrow aspirate samples from all 35 horses were analyzed using flow cytometry. In a subpopulation of horses (n = 8), samples of bone marrow aspirate before and following density gradient centrifugation were submitted for cytological analysis.
Samples from some horses (n = 14) were used only for antibody validation and were not cultured. The remaining samples (n = 21) were subsequently cultured as described below. Bone marrow samples from a subpopulation (n = 6) of horses were cultured for 14 days and then utilized for analysis of DNA content to determine the cell cycle state. Some samples (n = 6) did not have sufficient cell numbers to complete analysis at all time points; yet they were used for flow cytometry at one or more culture time points to check for repeatability or alterations in cell surface protein expression. Samples from three horses were cultured for 21 days and then subjected to tri-lineage differentiation assays. Sufficient cell numbers for protein and gene expression analysis at all time points were available from six horses.
MPC expansion in culture
BMMNCs were plated onto 10 cm diameter tissue culture plates at a density of ~300,000 cells/cm2 (20 × 106 cells/plate). Cells were cultured at 37°C in a 5% CO2, 95% air atmosphere at 5% humidity. Cells were cultured in media containing Dulbecco’s modified Eagle’s medium (DMEM, glucose at 1,000 mg/L), 2 mM l-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL), basic fibroblastic growth factor (bFGF, 1 ng/mL), and 10% fetal bovine serum. One-half of the media (5 mL) was removed at 24 h of culture and replaced with fresh media. Subsequently, media were exchanged every 72–96 h. At subconfluence of 70%–90%, cells were passaged 1:3 using Accumax® cell dissociation solution (Innovative Cell Technologies Inc, San Diego, CA) and plated at a density of about 10,000 cells/cm2. Approximately 10 × 106 cells from each sample were analyzed by flow cytometry for cell surface protein expression at 2 h and on days 2, 5, 7, 14, 21, and 30 of culture. Cells were analyzed at these time points to evaluate the changes in cell surface proteins over time, and to characterize the cells prior to performing differentiation assays.
Flow cytometry analysis
Cell surface markers of putative stemness were assessed using flow cytometry. Cells were pelleted in aliquots containing 1 × 106 cells and labeled for cell surface molecules selected from a panel of monoclonal antibodies known to define human MPCs (). Cells were treated with a 20-min blocking step using 10% normal goat serum in FACS-Buffer (phosphate-buffered saline containing 2.5% fetal bovine serum). The cells were pelleted, washed with FACS-Buffer, and pelleted again. Cell pellets were resuspended in fluorescent-conjugated or -unconjugated primary monoclonal antibody and incubated for 45 min at 4°C. Cells were then washed, a second fluorescent-conjugated goat anti-mouse IgG or IgM antibody (fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) or IgM μ-Chain-Specific, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was applied to the unconjugated antibodies, and the samples were incubated for an additional 45 min at 4°C. The CD29 antibody was directly conjugated with PE (read at FL2); all others were labeled with FITC-conjugated secondary antibody (read at FL1). Cells were resuspended in FACS-Buffer and analyzed on a FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) flow cytometer equipped with a 488 μm argon laser and BD Cell Quest™ analysis software (BD Biosciences, San Jose, CA). Cells not treated with antibody, and cells exposed to mouse anti-parvovirus antibody and FITC or PE-conjugated secondary antibodies were used as negative controls. The settings for the flow cytometric analyses determined <2% positive cells for the control antibodies. Data were collected on 1 × 105 cells for each sample regardless of size and granularity to prevent bias in gating.
For culture-expanded cells, flow cytometry analysis was performed on days 2, 7, 14, 21, and 30 following isolation. Supernatant was removed and adherent cells were lifted from the plate using Accumax® solution (1 mL/15 cm2) to prevent damage to cell surface proteins and avoid cellular clumping. Cells were processed and analyzed by flow cytometry as described earlier, except dot plot settings were adjusted to a logarithmic scale in the cultured cells to include large, granular cells. Flow cytometric analysis of cell surface molecule expression was performed in the gate determined to contain dividing cells based on the results from the propidium iodide DNA staining assay described later.
Propidium iodide DNA staining assay for cell cycle analysis
Propidium iodide can be used to determine DNA content in cells and identify populations of cells undergoing division. A reported feature of MPCs is their ability to proliferate [2
]. To determine the region of cell division, samples (0.5 × 106
cells) from six cultures were collected on Day 14 and resuspended in 500 μL hypotonic propidium iodide solution containing 0.05 mg/mL propidium iodide, 1 mg/L sodium citrate, and 0.1% Triton X-100 [16
]. Samples were protected from light and incubated at 4°C until analysis. Samples were analyzed by flow cytometry on FL2. Histograms were plotted for each cell population on a linear scale. The DNA content is proportional to the mean fluorescence intensity, and consequently indicates the stage of cell division (G0/G1, S, G2, M; cells in subG0 are dead) [17
RNA extraction and one-step reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
Gene expression analysis was included to confirm negative protein results and account for kinetic changes in transcription and translation. At the same time points when cells were analyzed by flow cytometry, RNA was extracted from ~1–3 × 106 cells of the corresponding samples using Trizol® (Life Technologies, Invitrogen, Carlsbad, CA) according to the manufacture’s directions. RT-qPCR was performed to provide supporting evidence that gene expression levels were consistent with cell surface protein expression levels. RNA quantity and quality were determined using a Nanodrop® spectrophotometer (NanoDrop Technologies, Inc, Wilmington, DE), and visualization of 18 and 28S bands on 0.8% agarose gels. Gene segments were cloned and novel sequence data files were submitted to Genbank (accession numbers, EF442070 for CD13; EF442071 for CD29; EF576851 for CD45; EU881920 for CD90; and EU881921 for CD11a). A portion of the CD44 gene was also cloned and agreed with previously reported data (X66862).
Total RNA was reverse-transcribed and amplified using the one-step RT-PCR technique and the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). The primers and dual-labeled fluorescent probe [6-FAM as the 5′ label (reporter dye) and TAMRA as the 3′ label (quenching dye)] were designed using Primer Express Software Version 2.0b8a (Applied Biosystems, Foster City, CA). All probes and primers were designed using equine-specific sequences published in Genbank, or sequenced in our laboratory (). Since several isoforms (five in humans) of CD45 exist, primers and probes were designed to detect as many equine isoforms as possible (equivalent to four of the five human isotypes). Two genes (CD45 and CD11a) did not reach a C
T value in later time point samples. Therefore, normalized copy numbers per nanogram of RNA values for each gene were calculated. A quantity value of 1 was assigned to samples that did not reach a C
Primers and Probes Utilized in RT-qPCR of Mesenchymal Progenitor Cell Marker Genes
MPC differentiation assays
To verify that cultured bone marrow cells were capable of tri-lineage differentiation, 10 × 106 culture-expanded cells from three horses were used for adipogenic, osteogenic, and chondrogenic induction assays.
Aliquots of MPCs (0.2 × 104
cells/well) were treated with 5% rabbit serum (lot 24129, Innovative Research, Novi, MI) in culture media to induce adipogenesis [18
]. Media was changed at Day 4 following induction. Samples were collected on days 1, 3, and 7 post-induction. To assess adipogenic differentiation, cells were fixed in 4% paraformaldehyde, incubated in a solution containing Oil-Red-O for 10 min to stain for lipid inclusions, and counterstained with hematoxylin. Stained samples were imaged using standard microscopy and graded positive or negative for Oil-Red-O staining.
Osteogenic induction. Aliquots of MPC (0.2 × 104 cells/well) were treated with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid (all Sigma-Aldrich, Inc., St. Louis, MO) in low glucose DMEM/10% fetal bovine serum media. Media was changed at Day 4 for the 7-day culture samples. Samples were collected on days 1, 3, and 7 following induction to assess early osteogenic differentiation. To assess calcium accumulation, cells were fixed in 4% paraformaldehyde and incubated in 2% aqueous Alizarin Red S (Sigma) for 3 min, followed by counterstaining with hematoxylin. Stained samples were imaged using standard microscopy and assessed for Alizarin Red staining. Intracellular calcium concentration was measured using a commercially available kit (QuantiChrom™ Calcium Assay Kit, BioAssay Systems, Hayward, CA) in cell extracts collected on days 2, 3, 4, and 7 following induction. Protein content in the same cell extracts was determined using the Bradford protein assay kit (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as the standard. Calcium concentration was expressed as μg Ca2+/μg of total protein.
Pellet cultures were generated using 5 × 105
cells/pellet with processing as previously described [19
]. Pellet cultures were maintained in medium consisting of high glucose DMEM containing 100 μg/mL sodium pyruvate, 10 ng/mL TGF-β3, 100 nM dexamethasone, 1× insulin/transferrin/selenium (ITS+1) premix, 40 μg/mL proline, and 25 μg/mL ascorbate-2-phosphate (all Sigma-Aldrich, Inc., St. Louis, MO). Medium was replaced twice weekly. Samples were collected on days 1, 3, 7, 14, 21, and 28 of culture. Pellets were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 4 mm sections. Matrix metachromasia was assessed with Safranin-O/fast green staining.
Gene expression data were categorized into four groups by culture duration: 1 = <1 week; 2 = 1 week; 3 = 2 weeks; 4 = 3 weeks or more. Calcium/protein ratio data were categorized into one of four groups by induction duration: 1 = control (no osteogenic induction); 2 = 48 h of induction; 3 = 72 h of induction; 4 = 1 week of induction. Groups were compared using a one-way ANOVA with a Tukey all-pairwise comparisons post hoc test. A P value of <0.05 was considered significant.