Introduction
Mesenchymal stem cells (MSCs) are a population of adult stem cells harboring an important differentiation potential, which can be utilized for tissue regeneration though the exact nature remains elusive [1, 2]. Dental pulp stem cells (DPSCs) are a unique type of MSCs residing in the pulp tissue of teeth [3]. As MSCs can be found in bone marrow, dental pulp shelters DPSCs. Dental pulp and bone marrow shares similarities such as surrounding by mineral tissue, innervation, and high vascularization. DPSCs are involved in maintaining tooth homeostasis and repairing damaged dentin through the generation of odontoblasts [4]. Lineage tracing in mouse incisor showed that cells from vascular origin actively contributed to injury repair [5]. Granting DPSCs are perfect candidates in the development of therapeutic resources in the regenerative dentistry, the characteristics of DPSCs have not been fully evaluated [6-8].
Tissue-resident adult stem cells, including DPSCs, have the abilities to self-renew and generate the differentiated cell types present in the tissues. In spite of the potential in tissue regeneration, the study of organ development and tissue patterning in vitro using stem cells has been delayed due to the lack of appropriate culture system that recapitulates the three-dimensional (3D) interactions necessary for organ morphogenesis. Organoid is a 3D cell mass containing stem cells and neighboring cells, in which cells are self-organized in a way similar to in vivo counterpart [9, 10]. Organoids can be derived from adult tissue pieces harboring stem cells, differentiating embryonic/pluripotent stem cells, or even a single adult stem cell [11]. Organoids can be utilized for understanding the characteristics of stem cells, the development of tissues during embryogenesis and the regeneration processes in the adult tissues, and for developing regenerative therapeutics by providing a platform for drug screening or transplanting organoids to damaged tissues [12]. In oral biology, the progresses in generating salivary gland organoids or organ germs have lead the emergence of bioengineered glands [13, 14]. However, organoids originated from various dental stem cells, including DPSCs, have not been generated.
In the present study, we subjected dental pulp cells isolated from dental pulp tissues (DPCs) and DPSCs to 3D organoid culture and evaluated the differentiation potential. We observed that DPCs and DPSCs in 3D organoid culture were differentiated into vascular endothelial cells with tube forming activity. These observations may provide a novel utility of DPSCs and a platform to identify the origin of DPSCs in dental pulp tissue.
Materials and Methods
Cell culture
Proietics™ human Dental Pulp Stem Cells (DPSCs) were from the third molar of an anonymous adult male donor and cryopreserved at a primary passage (PT-5025, Lonza). These cells are positive for CD105, CD166, CD29, CD90, and CD73, negative for CD34, CD45, and CD133. DPSCs were maintained and expanded in Miltenyi Stem MACS MSC Expansion Media Kit XF (“StemMacs”; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with 100 U/mL penicillin-G, 100 μg/mL streptomycin in 15 cm cell culture plates. At passage completion, cells were detached from culture plates using TrypLE™ Express (Life Technologies) for 3 min and replaced at a density of 5000-6000 cells/cm2.
Isolation and culture of dental pulp stem cells
Two molar teeth were obtained from one male (age 17 years, DPCs#1) and one female (age 24 years, DPCs#2) donors. Immediately after extraction, the teeth were placed in basic medium (Dulbecco’s modified Eagle’s medium, DMEM), transported to the laboratory, and washed with phosphatebuffered saline (PBS, Invitrogen, Carlsbad, CA, USA). The tooth surfaces were cleaned and the pulp chamber was revealed by cutting around the cementoenamel junction with sterilized dental fissure burs. The pulp tissue was gently separated from the teeth and divided into fragments approximately 1 mm × 1 mm × 2 mm in size. DPCs were then isolated and cultured by MSC-EM the dental pulp tissue was digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase (Sigma, St. Louis, MO, USA) for 30 - 60 minutes at 37℃. DPCs were obtained by passing the digested tissue through a 70 μm cell strainer (Falcon, BD, Franklin Lakes, NJ, USA). Single cell suspensions (1 × 105cells/flask) were seeded in MSC-EM supplemented with 100 U/mL penicillin-G, 100 μg/mL streptomycin, and 0.25 μg/mL Amphotericin B (Fungizone; GIBCO, Grand Island, N.Y.). Cells were maintained at 37℃ in a 5% CO2 atmosphere.
Organoid culture of DPCs or DPSCs
The culture of long-term expandable DPSCs organoids allows the establishment of organoid lines to investigate aspects of dental biology in vitro. The DPCs suspension was filtered through a 70-μm cell strainer into a 50 ml conical tube. The filter was rinsed with 5 ml of cold PBS buffer. Samples were visualized again and a portion of the suspension was centrifuged for 5 minutes at 4°C. Supernatant was gently poured off, ensuring all excess liquid was removed from the tube. DPCs and DPSCs were resuspended in Cultrex® RGF BME Type 2 (BME 2) plus the organoid growth media (OGM) and then plated onto 60mm Nunclon™ Sphera™ Dishes. OGM was replaced every 3–4 days.
Flow cytometry analysis
Analysis of fluorescence intensity of the stained cells was performed using a FACSCanto flow cytometry system (BD Biosciences). DPCs and DPSCs were dissociated with TrypLE ™, the cell pellet resuspended in PBS with 1% FBS and aliquot at 1 × 105 cell/tube for antibody labeling. Cells labeled with CD31 antibodies were analyzed by counting 10,000 events. 7-AAD was added at a 1:100 dilutions to distinguish live cells from dead cells. Obtained fluorescence signals were analyzed using the FACSDiva (ver 6.1.3, BD Biosciences) or the FlowJo (ver 10, Tree Star Inc.).
Tube formation assay
For tube formation assay of DPCs, aliquots (35 μl) of growth factor-reduced Matrigel™ (BD Biosciences, San Jose, CA, USA) (10 mg protein/ml) were added to 96-well culture dishes and polymerized for 30 min at 37°C. DPCs were trypsinized, resuspended in Stem MACS MSC Expansion Media and IntestiCult™ Organoid Growth Medium, and plated onto a layer of Matrigel at a density of 1 × 104 cells/well. After incubation of the Matrigel cultures at 37°C overnight cells were labeled with 3 μM Calcein AM for 30 minutes at 37°C in 5% CO2 incubator. The cultures were photographed using 484 nm excitation and 520 nm emission filter on a fluorescent microscope equipped with × 10 objective [15]. The images of the tubes were scanned into Adobe Photoshop (version 7.0.1) and quantified using ImageJ software (ver 1.52c, National Institutes of Health).
Immunofluorescence staining
For immunofluorescence staining, cells were fixed in 4% paraformaldehyde in PBS for 10 min, washed twice with PBS, and blocked with 1% FBS in PBS for 30 min; all procedures were performed at room temperature. The fixed specimens were incubated with primary antibodies for 1 h, followed by incubation with secondary antibodies for 1 h. Primary antibodies (1:100) were detected by Alexa Fluor 488 and Alexa Fluor 568 conjugated secondary antibodies (1:1000) (Invitrogen, CA). The specimens were finally washed and mounted in Vectashield medium (Vector Laboratories, CA) with 4',6-diamidino-2- phenylindole for visualization of nuclei. The stained sections were visualized using laser scanning confocal microscopy (Olympus FluoView FV1000).
Statistical analysis
Data are expressed as mean ± S.E. for in vitro studies. Statistical significance (p<0.05) was determined using two tailed unpaired t-tests. Unless stated otherwise, all experiments were performed in triplicate.
Results
Human dental pulp stem cells in organoid culture
DPCs were isolated after 2D plating of human dental pulp extract from the molar and expanded successfully in MSC-EM. The culture showed the cell proliferation in 3 days after the initial plating. Cells exhibited homogenously fibroblast-like morphology. We attempted to establish dental pulp organoid culture by subjecting 2D-expaned DPCs to a 3D extracellular matrix (BME2) culture with OGM (Fig. 1A). DPSCs along with DPCs were also subjected to the organoid culture in comparison (Fig. 1B). Organoid culture condition for intestinal stem cells was adopted, and cells were effectively expanded.
Tube formation during organoid culture
DPCs cultured for 7 days in MSC-EM were reseeded into BME2 dome matrix with addition of OGM. After 17 hours in the organoid culture condition, cells showed morphological changes inside BME2 matrix, which resulted in endothelial progenitor cell appearance and tube forming activity (Fig. 2). DPSCs followed the same procedure and showed the endothelial cell-like morphological changes and tube forming activity inside BME2 matrix. During this short period of organoid culture for 17 hours, DPCs and DPSCs transformed to endothelial cell-like status. However, tube forming activity was transient in DPCs#2 and DPSCs and eventually tubes disappeared after 18 hours. Interestingly, DPCs#1 showed the persisting tube forming activity. These results suggest that dental pulp-derived cells are converted to endothelial-like cells with tube forming activity in the organoid culture but the persistence of tube formation varies.
Tube formation assay with organoid culture-derived cells
To verify the tube forming activity of dental pulp-derived cells in the organoid culture, DPCs and DPSCs inside BME2 in OGM were harvested and subjected to tube formation assay. As microscopic observation showed the evident tube forming activity during 3-17 hours after reseeding in the organoid culture, cells were harvested at 17 hours. DPCs and DPSCs maintained in MSC-EM were also subjected to tube formation assay in comparison. Calcein staining showed that cells maintained in MSC-EM did not have tube forming activity. However, cells harvested from the organoid culture showed the high activity of tube formation (Fig. 3A). DPCs#2 showed a relatively low activity in tube formation probably due to the relatively-late time point of harvesting. Measuring tube length showed a significant increase in DPCs#1 and DPSCs harvested from the organoid culture (Fig. 3B). These results suggest that dental pulp-derived cells exposed to the organoid culture for a short period time exhibited a high activity in tube formation.
CD31 expression in organoid culture-derived cells
To evaluate the expression of CD31, endothelial cell marker, on organoid culture-derived cells, DPCs#1 and DPSCs harvested from BME2 in OGM at 17 hours after reseeding were subjected to immunocytochemistry and flow cytometry analysis. Immunocytochemistry analysis along with DAPI staining showed that DPCs#1 and DPSCs harvested from the organoid culture expressed CD31 (Fig. 4A). When cells from the organoid culture were subjected to flow cytometry analysis in comparison with cells maintained in MSC-EM, DPCs#1 and DPSCs showed a significant elevation of CD31 expression on the surface in comparison with cells maintained in MSC-EM (Fig. 4B). These results suggest that dental pulp-derived cells undergo endothelial phenotypic and functional changes during the early phase in the organoid culture.
Discussion
Different types of adult stem cells have been identified in the human body. These adult stem cells have known to be involved in tissue homeostasis or damage repair [16]. Dental stem cells are recently gaining attention due to their versatility in tissue repair beyond the dental structures [4, 17]. The advantages of dental stem cells over other adult stem cells include the relative availability of the source tissue and the access to the donors. Among various dental stem cells, DPSCs have shown the excellent differentiation potential and the application to tissue repair [3]. Despite the increase interest in dental stem cells, the characteristics of DPSCs have not been fully understood. Thus, we evaluated the cellular behavior of dental pulp-derived cells in the organoid culture.
Organoid culture is an excellent system for identifying and characterizing adult stem cells as it provides a long term culture in which adult stem cells self-renew, differentiate, and form a 3D structure recapitulating in vivo counterpart [10, 11]. Organoid culture can be established from crude tissue extracts or a single stem cell. When we first subjected the crude fragments from dental pulp after chopping to organoid culture, it was difficult to identify growing colonies due to high background. For the next step, we established a 2D culture of dental pulp cells (DPCs) and subjected them to organoid culture. Interestingly, at the early phase of organoid culture, tube formation was observed inside the extracellular matrix dome. To compare whether this phenotypic changes are from DPSCs, we subjected commercially-available DPSCs to organoid culture and observed the same result. Regarding the report proposing the perivascular origin of DPSCs, DPCs and DPSCs in our culture may harbor perivascular sources [5]. To clarify the differences among DPSCs and DPCs, the analysis of the bigger size samples may be necessary.
Vascular activity of dental pulp-derived cells was rather transient in the organoid culture. We utilized the organoid culture method for intestinal stem cells [18]. Transient vascular activity could be related to a suboptimal culture condition for dental organoids. On the contrary, the vascular activity could have been resulted from the interaction between pulp cells and endoderm cell culture media as observed during embryo development [19]. Another origin of DPSCs was demonstrated as neural crest cell-derived Schwann cells [8]. It is interesting to note that DPSCS can contribute to neural tissue repair [17]. Althoug DPSCs and DPCs showed CD31 expression, other markers may need to be tested to clarifiy the identity. The exact nature of DPSCs remains for the future study.
In conclusion, we demonstrated the effect of OGM culture of dental pulp-derived cell on the promotion of endothelial cell fate and function. The potential of DPSCs as a therapeutic source or a screening platform for the development of regenerative medicine has been increasing. Accurate understanding of populations and characteristics comprising DPSCs will expedite the practical outcome and the clinical application. We expect the combination of organoid culture and various cellular analyses will lead to the true identity of DPSCs.
