Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs
- Brooke R Snyder†1, 2,
- Pei-Hsun Cheng†1, 2,
- Jinjing Yang1, 2,
- Shang-Hsun Yang1, 2,
- Anderson HC Huang3 and
- Anthony WS Chan1, 2Email author
© Snyder et al; licensee BioMed Central Ltd. 2011
Received: 10 June 2011
Accepted: 12 September 2011
Published: 12 September 2011
Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated.
To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile.
Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.
DPSCs are ASCs and were first described in 2000 . DPSCs share similar characteristics with bone marrow derived mesenchymal stem/stromal cells (BMSCs) [1–4]. DPSCs have been differentiated into neuronal cells under the guidance of neurogenic factors [5–10]. Additionally, DPSCs may also be therapeutic by providing trophic support [4, 11] or recruiting endogenous cells for repair [4, 12].
Most studies involving ASCs are derived from healthy donors, and studies involving DPSCs have, in general, been limited. Due to the advantages of using one's own cells for therapy [13, 14], plus their potential use in the central nervous system (CNS) [4–9, 15, 16], we are interested in characterizing DPSCs derived from individuals with HD and determined if HD-DPSCs retain comparable stem cell properties to those derived from a healthy individual.
HD is a dominant genetic disorder caused by a mutation resulting in the expansion of polyglutamine (CAG) repeats in exon 1 of the IT15 gene encoding for htt. CAG repeat lengths over 39 results in pathological HD. A negative correlation has been shown between repeat length and age of onset  and lifespan . The hallmark of HD is neurodegeneration, predominantly in the striatum and cortex . Many HD patients do suffer from tooth decay; however this decay is due to medication and lack of motor skills to perform teeth maintenance procedures [20, 21]. Here we have used transgenic HD monkeys  as a model for investigating the effect of mutant htt on the properties of DPSCs.
Establishment of DPSCs from HD monkeys
Expression of stem cell specific transcription factors in rHD/G-DPSCs
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  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].
Cell surface antigen profile
Cell surface antigen profiles of ASCs derived from monkey, chimpanzee and human
Expression of mutant htt in rHD/G-DPSCs
Latest advancements in therapeutic applications of ASC have driven our interest in evaluating a patient's own DPSCs as an alternative source for cell therapy. Although recent studies have demonstrated the differentiation potential of DPSCs toward neuronal lineage [5, 6], in-depth characterization such as biofunctions in vivo will further determine the therapeutic potential of DPSC derived neural cells. While therapeutic applications of DPSCs in cell replacement therapy are at preliminary stage, continued development in differentiation protocol and in vivo validation are important steps. Unlike cell replacement, endogenous stem cells are expected to divide and differentiate under the influence of the DPSC graft and the microenvironment [4, 5, 12].
In this study, HD-DPSCs were derived from teeth buds of miscarried HD monkeys and HD monkeys that died soon after birth. While the biofunctions of HD-DPSCs may vary from those derived from adult individuals, our findings suggested rHD/G-DPSCs retain properties comparable to ASCs [3, 4, 24, 27]. Additionally, the impact of age on the biofunctions of DPSCs has not been fully addressed and it is important for determining future applications of DPSCs derived from individuals at different ages. Although we speculate the biofunctions of DPSCs will not be impacted significantly by age, the derivation efficiency of DPSCs may vary due to the number of stem cells existed in the dental pulps. While the present study is aimed to determine if DPSCs with inherited genetic defect retain ASC properties, our future goal is to determine if DPSCs derived from living HD monkeys or patients can be used to derive neuronal cell types for replacement therapy. Moreover, genetic correction of HD-DPSCs may be considered prior cell transplantation because HD-DPSCs may not have longevity and may not function in the same way as DPSCs derived from normal animals.
Multipotent differentiation capability was evaluated by in vitro differentiation into osteogenic, adipogenic and chondrogenic lineages. Differentiation into these three lineages is considered one of the basic requirements for BMSCs, which is widely applied in ASCs . All rHD/G-DPSCs differentiated to osteogenic, adipogenic and chondrogenic lineages, which were comparable to rG-DPSCs and published reports (Figure 1) [2–4, 27, 28]. Recent studies have suggested that DPSCs are also capable of differentiating into neurons [5, 6, 8, 29]. Although the current paradigm of ASC therapy is primarily based on the homing nature of ASCs [12, 30, 31] and their capability of eliciting a local repair response [4, 12, 30, 32], their ability to differentiate into multiple cell types including neurons may be therapeutically relevant. While all tested cell lines were capable of differentiating into three different lineages, quantitative studies on specific lineage markers suggested variations in differentiation capacity among DPSC cell lines exists. Therefore, whether DPSCs derived from HD monkeys or normal rhesus monkeys have more preferred lineages upon differentiation cannot be concluded.
Our results suggested that DPSCs derived from HD monkeys retain ASC properties, thus one may consider that these cells also retain biofunctions comparable to those derived from healthy individuals . This study supports the potential future therapeutic application of DPSCs from patients with genetic disorders such as HD. We demonstrated that DPSCs from HD monkeys are comparable in all aspects that define ASCs. Thus DPSCs derived from patients with inherited genetics diseases hold great promise as an alternative source for personal cell therapy.
All transgenic HD monkeys were housed under the guideline of the IACUC approved procedures and the support of the Division of Animal Resources at the Yerkes National Primate Research Center (YNPRC). All procedures were approved by YNPRC/Emory Animal Care and Biosafety Committees.
Generation of transgenic monkeys 
Transgenic Huntington's monkeys were generated as described by Yang and colleagues . 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.
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.
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 
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 
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.
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.).
Data analyses were carried out using the Student t-test.
We thank Dimitri Fillos and Dr. Chris C. Ibegbu for their assistance in flow cytometry analysis. We also thank Dr. X.J Li for mEM48. All protocols involving animal care and handling were approved by Emory University's IACUC. Yerkes National Primate Research Center is supported by the base grant No.RR-00165 awarded by the NCRR/NIH. This study is supported by grant awarded by the NCRR/NIH (RR018827-04). The authors have no commercial association that might create a conflict of interest in connection with submitted manuscript.
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