Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs

Establishment of DPSCs from HD monkeys

We have isolated and established DPSCs from the teeth buds of three double-transgenic HD/G monkeys (rHD11, rHD17 and rHD18) and one transgenic GFP monkey (rGFP). rHD17 and rHD18 died shortly after birth at full term. Both rHD17 and rHD18 demonstrated HD phenotypes including severe dystonia and chorea [22]. rHD11 and rGFP were miscarried monkeys, born at four months of gestation. rHD/G-DPSCs have distinctive morphology including long and spindle shaped (Figure 1A) which were similar to published reports of rBMSCs [23], rhesus monkey DPSCs (rDPSCs) [4], Chimpanzee DPSCs (ChDPSCs) [2], hBMSCs and hDPSCs [3, 24, 25]. There were no overt differences in cellular characteristics of DPSCs derived from different individuals. All cell lines expressed GFP (Figure 1A) and were capable of self-renewal.

Expression of stem cell specific transcription factors in rHD/G-DPSCs

To further evaluate stem cell properties of rHD/G-DPSCs with various degree of mutant htt, quantitative measurement of stemness factors (Oct-4, Nanog and Rex-1; Figure 2) was performed by Q-PCR. There was no different in the expression pattern of stemness factors between canine and molar derived DPSCs. However, variations in expression levels were observed among individuals (Figure 2). rHD17 and rHD18 have lower expression compared to rHD11 and rGFP.

Differentiation competence of rHD/GFP-DPSCs

One of the defining characteristics of ASCs is multipotent differentiation capability. All rHD/G-DPSCs were capable of differentiating into osteogenic (Figure 1B), adipogenic (Figure 1C) and chondrogenic lineages (Figure 1D), which are trademark events of ASCs [24, 26] and DPSCs [3, 4]. The multipotent differentiation capability of rHD/G-DPSCs and r/G-DPSCs were similar to published reports of rhesus macaques [4, 23], chimpanzee [2] and humans [3, 24] ASCs. Similar to previous studies, the fat droplets in DPSCs were relatively smaller and less intense than that of BMSCs [2, 4].

The effect of mutant htt on the differentiation capacity of HD-DPSCs was further determined by quantitative measurement on the expression of lineage specific markers including osteopontin (osteogenic marker), lipoprotein lipase (adipogenic marker) and collagen II (chondrogenic marker) by Q-PCR (Figure 3). There was no detectable expression of lipoprotein lipase and collagen II prior to induction. On the other hand, osteopontin was expressed before induction and was increased in some of the DPSC lines after induction. Variations among cell lines were also observed (Figure 3).

Cell surface antigen profile

Expression of mutant htt in rHD/G-DPSCs

In order to determine the effect of mutant htt on the properties of rHD/G-DPSCs, the expression of mutant htt and the formation of oligomeric htt were evaluated. Expression of mutant htt was significantly increased in all HD-DPSCs determined by Q-PCR (Figure 4A). Among HD-DPSC lines, rHD17 had the highest expression of mutant htt, while rHD11 has the lowest (Figure 4A). The formation of mutant htt aggregate was revealed by using western blotting analysis (Figure 4B) and immunostaining (Figure 4C) using mEM48, a monoclonal antibody that binds to human htt enhanced with a polyQ expansion. Nuclear inclusions and mutant htt aggregates were observed in all rHD/G-DPSCs, but not in rG-DPSCs (Figure 4C). Among the three HD monkeys, rHD17 had the most extensive intranuclear inclusions and cytoplasmic mutant htt aggregates compared to rHD11 and rDH18. Western blotting analysis further confirmed the presence of oligomeric htt at a high molecular weight (> 250 kD) in the upper portion of a gradient polyacrylamide gel (Figure 4C) in all HD-DPSC cell lines. Again, rHD17 had a more intense band than rHD11 and rHD18, suggesting the presence of more oligomeric htt. The observation of htt by immunostaining of rHD/G-DPSCs was consistent with the expression level of mutant htt determined by Q-PCR and the extent of oligomeric htt demonstrated by Western blot analysis.

Generation of transgenic monkeys [22]

Transgenic Huntington's monkeys were generated as described by Yang and colleagues [22]. In brief, lentiviruses carrying the Exon 1 of the htt gene containing expanded CAGs under the control of ubiquitin promoter were used to infect metaphase II arrested rhesus monkey oocytes followed by fertilization and embryo transfer into surrogate females.

Isolation and culture of DPSCs [4, 25]

The teeth germs/buds were recovered from monkeys miscarried at four months gestation (rHD11 and rGFP) and died soon after birth (rHD17 and rHD18). The teeth germs/buds were then digested in 3 mg/ml collagenase type I and 4 mg/ml dispase (Invitrogen, Inc) for one hour at 37°C. Single cell suspension was filtered through a 70 μm cell strainer and was then cultured in DPSC culture medium (α-MEM (Invitrogen, Inc) supplemented with 20% FBS (Atlanta Biologicals, Inc), 2 mM glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Inc.)) at 37°C with 5%CO₂.

Adipogenic, osteogenic and chondrogenic differentiation [2, 4, 25]

For adipogenic differentiation, cells were seeded at 400 cells/35 mm tissue culture dish and cultured for 11 days in DPSC medium. On day 11, the medium was supplemented with 5.0 μg/ml insulin, 50 μM indomethacin, 1 μM dexamethasone, and 0.5 μM IBMX, which was replaced every 3-4 days for a total of three weeks. The culture was then fixed in 4% paraformaldehyde (PFA) and stained with 0.0125% Oil-Red-O in isopropanol for 20 minutes at RT followed by a thorough wash and microscopic examination.

For osteogenic differentiation, cells were prepared as described for adipogenic differentiation until day 11. On day 11, the medium was supplemented with 1 nM dexamethasone, 50 uM L-Ascorbic acid 2-phosphate sesquimagnesium salt, 20 mM β-glycerolphosphate, and 50 ng/ml L-thyroxine sodium pentahydrate, and was replaced every 3-4 days for a total of three weeks. The culture was then fixed in 4% PFA and stained with 1% Alizarin Red S, pH 4.1 for 20 minutes at RT followed by a thorough wash and microscopic examination.

For chondrogenic differentiation, 2.5 × 10⁵ DPSCs were centrifuged in a 15 ml conical tube at 1,000 rpm for five minutes. The pellet was maintained in a DPSC medium supplemented with ITS-plus premix (BD Biosciences) to a final concentration of 6.25 ug/ml insulin, 6.25 ug/ml transferrin, and 6.25 ng/ml selenious acid. Additionally, 5.35 ug/ml linoleic acid, 1.25 mg/ml bovine serum albumin, 50 μg/ml Ascorbate 2-phosphate, 40 μg/ml L-proline, 100 μg/ml Sodium pyruvate, 100 nM Dexamethasone, 100 units/ml penicillin, 100 μg/ml streptomycin and 10 ng/ml TGF-β3 (R&D Systems) were also supplemented. Medium was replaced every 3-4 days for a total of four weeks. The pellets were then fixed in 4% PFA overnight. The paraffin-embedded sections (4-5 μm) were stained with 1% Alcian blue in 10% sulphuric acid solution for 15 minutes followed by a thorough wash and microscopic examination.

Quantitative Real-Time PCR (Q-PCR)

RNA from cell samples was prepared using RNeasy Mini kit (Qiagen). An equal amount of total RNA was used to synthesize cDNA followed by Q-PCR using iQ5 Real-Time PCR Detection System (Bio-Rad). Specific-qPCR primer sets targeting stem cell and differentiation markers were used (Additional file 1).

Immunocytochemistry and fluorescent microscopy [22]

Cell samples were fixed using 4% PFA, permeabilized and blocked. The expression of mutant htt was then incubated with mEM48. The mEM48 (1:200) immunoreactive product was visualized with the avidin-biotin complex kit (Vector ABC Elite). For fluorescent microscopy, the samples were examined with an Olympus BX51 epifluorescent microscope.

Western Blotting [22]

Total protein was extracted and concentrated for analysis using the Bradford Assay (Bio-Rad, Inc.). Equal amounts of the protein were boiled prior to polyacrylamide gel electrophoresis, the proteins were transferred onto a PVDF membrane (Millipore Immobion P, Millipore, Inc.) using Bio-Rad's transblot. The membrane was then blocked, incubated with primary antibody, secondary antibody, and detected using Amersham's ECL kit (Amersham, Inc.). The amount of protein was quantified using a densitometer.

Flow cytometry analysis [2, 4]

Cell samples at 2 × 10⁵ cells/tube were stained with FITC or PE-conjugated anti-CD14, -CD45, -CD59, -CD73, -CD90, -CD150, -CD166, -IgG1k, -IgG2ak (BD Pharmingen), or anti-CD18, -CD24, -CD29, -CD34, -CD44 (BD Biosciences), or anti-CD105 (eBioscience). After incubating 20 minutes at RT in the dark, cells were washed with 2 mL FACS wash solution (dPBS+1%BSA+0.1%NaN₃) and centrifuged five minutes at 230 × g. Supernatant was removed and cells were fixed with 1% formaldehyde. All data was acquired using a FACS Calibur (Becton Dickinson) and analyzed using CellQuest (Becton Dickinson) and Flowjo software (Treestar, Inc.).

Statistical analysis

Data analyses were carried out using the Student t-test.