Introduction
Mesenchymal stem cells (MSCs) are adult stem cells residing in the organ-specific supporting tissue and regarded to contribute to the organ repair from the injury [1,2]. MSCs display the differentiation potential toward several lineages including adipocytes, osteoblasts, chondrocytes, and organ-specific adult cell types [3]. Dental pulp stem cells (DPSCs) are MSCs residing in dental pulp and can be differentiated to odontoblast and restore the damaged dentin [4,5]. Though the recent lineage tracing experiments and the single cell analysis revealed the developmental origin and the constituting cell types of dental pulp [6-8], the exact nature of DPSCs still remains elusive.
Organoid culture has been replacing the sphere culture, both of which have provided the platform for the long-term culture of adult stem cells [9]. In the sphere culture, serum-free minimal condition drives the formation of three-dimensional (3D) cell aggregates in the suspension resulting in the gradual increment of stem cell population during serial passaging whereas the heterogeneity and cellular arrangement of in vivo origin are well-preserved in the organoid culture [10]. Organoid is advantageous in studying the morphogenesis and the location of adult stem cells, but sphere culture provides the edge in enriching the stem cell pool for the further analysis [11]. As the major types of organoids have been derived from epithelial tissue, sphere culture may bridge the gap between the conventional two-dimensional (2D) culture and in vivo structurerecapitulating organoid culture [12].
Sox2 is a critical transcription factor in pluripotent stem cells and adult stem cells regulating self-renewal and differentiation [13,14]. Sox2 directs the differentiation and the maintenance of neural stem/progenitor cells [15,16]. Sox2 expression is well-documented in the epithelium of cervical loop in mouse incisor where dynamic stem cell activities are detected, but the expression in the dental pulp has not been reported [17]. In MSCs derived from human umbilical cord blood or bone marrow, SOX2 was shown to regulate stemness and differentiation especially at a low density [18,19]. In DPSCs, overexpression of SOX2 augmented the cellular proliferation, migration and adhesion, which were abolished by siRNA-mediated SOX2 knockdown [20].
In the present study, 3D culture, including organoid and sphere, of DPSCs were tested with an attempt to enrich and characterize the core-stemness population from the conventional culture. As a result, the sphere culture of DPSCs was established and exhibited the higher expression of SOX2.
Materials and Methods
1. Cell culture
Human DPSCs isolated from the third molar of an anonymous adult male donor and cryopreserved at a primary passage were purchased (PT-5025; Lonza, Basel, Switzerland). DPSCs were maintained and expanded in MSC expansion media (MSC-EM, Miltenyi Stem MACS MSC Expansion Media Kit XF; 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 TrypLETM Express (Life Technologies, Carlsbad, CA, USA) for 3 minutes and replaced at a density of 5,000–6,000 cells/cm2.
2. Isolation and culture of primary dental pulp stem cells
Molar teeth were obtained from a male (age 17 years) and a female (age 24 years) donors under IRB protocol (PNUDH-2020-003). The current study was performed in accordance with the gender equality guideline of International Journal of Oral Biology. Immediately after extraction, the teeth were placed in basic media (Dulbecco’s modified Eagle’s media, Gibco, Invitrogen, Carlsbad, CA, USA), transported to the laboratory, and washed with phosphate-buffered saline (PBS, Invitrogen). 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. Primary DPSCs (pDPSCs) were then isolated and cultured by MSC-EM after digestion of fragmented dental pulp tissue 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℃ and passing through a 70 mm cell strainer (Falcon; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Single cell suspensions (1 × 105 cells/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, NY, USA). Cells were maintained at 37℃ in a 5% CO2 atmosphere.
3. Organoid culture of primary dental pulp cells
Prior to establishing pDPSC culture, dental pulp cells (DPCs) freshly isolated from dental pulp were subjected to organoid culture by resuspending in Growth factor reduced Matrigel (GFR Matrigel, BD Biosciences, Bedford, MA, USA; Catalog No. 354230) and human organoid growth media (OGM, IntestiCult TM; STEMCELL Technologies, Vancouver, BC, Canada) followed by drop-plating onto 60 mm NunclonTM SpheraTM Dishes (ThermoFisher Scientific, Waltham, MA, USA). OGM was replaced every 3–4 days.
4. Sphere culture of DPSCs and pDPSCs
Single DPSCs or pDPSCs were resuspended in sphere culture media which consisted of the following: Neurobasal media (Life Technologies) supplemented with 20 ng/mL bFGF, 10 ng/mL EGF, 2.5 μg/mL amphotericin, 100 IU/mL penicillin, 100 μg/mL streptomycin, and B-27 Supplement (50×) (Life Technologies, without serum) in Ultra-Low Attachment sixwell plates. Fresh media was added every two or three days. Spheres were transferred to the next generation by dissociation into single cells with Accutase followed by filtering through a 40-μm cell strainer and plating at 104 cells/mL.
5. Immunofluorescence staining
For immunofluorescence staining, cells or spheres were fixed in 4% paraformaldehyde in PBS for 10 minutes, washed twice with PBS, and blocked with 1% fetal bovine serum in PBS for 30 minutes. All procedures were performed at 4℃ or room temperature. The fixed specimens were incubated with anti-SOX2 antibody (rabbit polyclonal antibody, Cat No. ab79351) or anti-Dentin sialophosphoprotein (DSPP) (mouse monoclonal, Cat No. sc-73632) at 4℃ overnight, followed by incubation with secondary antibodies at room temperature for 1 hour. Primary antibodies (1:100) were detected by Alexa Fluor 568-labeled goat anti-rabbit (1:1,000, Invitrogen, Cat No. A11011) or Alexa Fluor 488-labeled donkey anti-mouse secondary antibody (1:1,000, Invitrogen, Cat No. A32766). The specimens were finally washed and mounted in Vectashield medium (Vector Laboratories, Burlingame, CA, USA) with 4’,6-diamidino-2-phenylindole for visualization of nuclei. The stained sections were visualized using Invitrogen EVOSTM FL Auto 2 Imaging System.
6. RNA isolation and quantitative reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted using TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA) and reverse transcribed into cDNA using the Reverse Transcription cDNA Kit (#RT50KN; NanoHelix, Daejeon, Korea). cDNA in 1 μL of the reaction mixture was am-plified using the Ready-2×-Go pre-mix PCR kit (#PMD008L; NanoHelix) and 10 pmol each of sense and antisense primers. The thermal cycle profile was as follows: denaturation at 95℃ for 30 seconds, annealing at 54℃ for 30 seconds depending on the primers used, and extension at 72℃ for 30 seconds. Each PCR reaction was carried out for 25–30 cycles and PCR products were analyzed by 1% agarose gel electrophoresis. The following primer pairs were used: SOX2: 5′-CAACATGATGGAGACGGAGC- 3′, 5′-GTG CATCTTGGGGTTCTCCT-3′; GAPDH: 5′-TCACCATCTTCCAGGAGCG-3′, 5′-CTGCTTCACCACCTTCTTGA- 3′.
Results
1. Organoid culture of human DPCs
DPCs were isolated from human molars by mechanical dissection and enzyme digestion as single cells. Cell pellets were suspended and subjected either to 2D culture for the establishment of pDPSCs or to 3D organoid culture (Fig. 1A). In organoid culture (pDPC-OR), morphologically-distinct cell aggregates were identified within 3 days after initial plating. However, the formation of cell aggregates eventually disappeared in the following passaging, yielding non-proliferating individual cells (pDPC-OR1) (Fig. 1B). These results show the unsuccessful maintenance of DPCs in the organoid culture.
2. Sphere formation of pDPSCs in serum-free suspension culture
When DPCs from Fig. 1A were subjected to the conventional 2D-culture, adherent cells proliferated and pDPSC culture was established (pDPSC-AD). In the attempt to enrich the stem cell population in pDPSC-AD, cells were switched to serumfree minimal media. Spontaneous formation of compact multicellular sphere was observed on day 1 and spheres reached the maximum size on day 7 in the suspension culture (pDPSCSP) (Fig. 2). These results suggest that sphere-forming cells arise spontaneously from a minor population of pDPSCs and continue to grow as a sphere.
3. Sphere formation of conventional DPSCs
To address whether the difficulty in establishing organoid culture and the spontaneous formation of spheres from DPSCs are broadly applicable, conventional DPSCs purchased from Lonza were subjected to the organoid culture and to the sphere culture. As shown in Fig. 3A, DPSCs did not proliferate or generate a distinct 3D structure in the organoid culture (DPSCOR). DPSCs properly proliferated in the conventional 2D culture and started to form spheres as switched to serum-free culture condition. Small-size spheres appeared in the suspension on day 2–3 and continued to grow to become larger spheres. The average diameter of DPSC-SP was 130 μm. When spheres were passaged to the second round of sphere culture (DPSCSP1), numerous small-size spheres were generated (Fig. 3B). These results suggest that self-renewing spheres spontaneously arise from conventional DPSCs.
4. pDPSC Spheres and DPSC spheres express SOX2
When spheres generated from pDPSCs and conventional DPSCs were subjected to RT-PCR to examine the expression of stemness-related markers, SOX2 expression was significantly upregulated in spheres compared with adherent cells (Fig. 4A). Immunostaining of adherent cells and spheres confirmed the little expression of SOX2 in the adherent cell and the significant increase of SOX2 in the sphere of pDPSCs. DSPP expression also increased in the pDPSC-sphere compared with adherent cells (Fig. 4B). Spheres generated from conventional DPSCs showed the high expression of SOX2 (Fig. 4C). These results suggest that, though organoid culture was unsuccessful, stemness-high population can be enriched through the sphere culture of DPSCs (Fig. 4D).
Discussion
Sox2 expression has been well-documented in the oral epithelium of developing tooth and the dental epithelium at the cervical loop of mouse incisor [21]. Dental mesenchyme receives a stimulatory signal from the epithelium and develops to dental pulp wherein DPSCs reside [22]. Identification of SOX2 expression in spheres generated from pDPSCs and DPSCs was unexpected. Co-expression of DSPP and SOX2 may indicate the enrichment of DPSCs during the sphere culture. In the previous report, addition of Desert Hedgehog during the organ culture of mouse incisor pushed the expression of Sox2 from the outer enamel epithelium to the inner enamel epithelium and the transit amplifying cell zone in the pulp mesenchyme [23]. Another possibility includes the mesenchymal to epithelial transition of DPSCs toward epithelial phenotype. Examination of the differentiation potential of DPSC spheres toward odontogenic lineages or the increase of Sox2 expression in dental pulp during dentin-pulp damage repair may provide the further clue in the understanding of SOX2 function in DPSCs.
The 3D organoid culture was attempted with primary DPCs or conventional DPSCs, both of which resulted the unsuccessful establishment. Though cells were residing inside the extracellular matrix, the surrounding media was not optimized for DPSC culture. Replacing the current organoid media with MSC expansion media or starting with the intact dental pulp instead of single cells may lead to novel observations aiding the successful establishment of organoid culture with DPSCs. However, as organoid recapitulates in vivo 3D-cellular organization, the arrangement of niche and stem cells should be clarified by imaging in advance.
In conclusion, spheres were generated from adherent culture of DPSCs in the minimal media with increased expression of SOX2. Our study suggests that stemness-high population in DPSCs can be enriched by spheroid culture, which may bridge the gap between 2D culture and organoid culture in the pursuit for achieving regenerative treatment in dentistry.
