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치의과학 석사학위논문

Human Dental Epithelial Cells Induce

Odontogenic Differentiation of SHED

사람 치아 상피세포에 의한

치수 줄기세포의 상아모세포 분화 유도

2019년 2월

서울대학교 대학원

치의과학과 분자유전학 전공

채 근 영

Abstract

Human Dental Epithelial Cells Induce

Odontogenic Differentiation of SHED

Geun Young Chae

Molecular Genetics Major

Graduate School, Seoul National University

(Directed by Prof. Gene Lee)

Tooth organogenesis and regeneration occur through reciprocal interaction

between epithelial and ectomesenchymal stem cells. This cell-to-cell

communication is also a key regulator in the differentiation of ameloblasts

and odontoblasts which secrete enamel and dentin, respectively. In studying

tooth regeneration, the dental pulp has been widely used as the source of

mesenchymal stem cells. On the contrary, most of the epithelium is lost after

tooth eruption and root completion, and thus have not been vastly explored.

Recently, prior research has characterized dental epithelial cells known as

Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM)

extracted from human periodontium and its cell line was established.

However, the epithelial-mesenchymal signaling capacity of HERS/ERM has

yet to be elucidated. Thus, this study was conducted to elucidate the effect of

HERS/ERM conditioned medium (CM) on the odontogenic differentiation of

stem cells from human exfoliated deciduous teeth (SHED). To simulate the

effect of epithelial paracrine actions on mesenchyme, SHED were cultured in

differentiation medium supplemented with varying proportions of CM

derived from the HERS/ERM cell line. The CM collected was freeze-dried

for further concentration of the solution, and the potency of CM was tested at

1X (differentiation medium supplemented with 10 v/v% CM), 4X and 8X

concentration factors, using freeze-dried CM for the 4X and 8X treatments.

To determine the effects of the varying concentration of CM, Alizarin red S

staining, RT-qPCR, Western blot, and DAPI nuclear staining were assessed

and the results were compared to SHED treated with the freeze-dried basal

media of CM. Alizarin red S staining revealed that increasing the

concentration of CM treatment had a distinctive impact on the amount of

calcium nodule formed in the differentiated SHED. Expression levels of

mineralization-related markers, RUNX2, BSP, DMP1, ON, OC, and MEPE

also confirmed the enhanced odontogenic effect of CM concentration on day

8 and 12 as CM concentration was increased. In contrast, the addition of

freeze-dried basal media exhibited a lack of calcium nodule formation and no

significant changes in the mineralization-related mRNA expression levels.

The expression of dentin phosphoprotein, an odontoblast marker observed by

Western blot, was also more prominent in CM-treated SHED than in basal

media-treated SHED on both day 16 and 20. Also, long-term culture of SHED

with CM exhibited cell death, whereas the basal media control group did not.

The DAPI nuclear staining revealed a phenomenon similar to the terminal

differentiation of cells. The data indicate that CM from human HERS/ERM

cell line has odontogenic differentiation capabilities that are concentration-

dependent, and further investigations may contribute to the discovery of

specific growth factors and cytokines at play. This study is the first report of

odontogenic induction potential of concentrated CM derived from the human

dental epithelial cell line.

………………………………………

Keywords : Hertwig’s epithelial root sheath/epithelial rests of Malassez

(HERS/ERM), stem cells from human exfoliated deciduous teeth (SHED),

epithelial-mesenchymal interaction, conditioned medium, tooth

development and regeneration, odontogenic differentiation

Student Number : 2017-24083

4

Tables of Contents

Introduction .............................................................................................. 5

Literature Review ..................................................................................... 7

Materials and Methods ............................................................................. 14

Results ...................................................................................................... 20

Figures and Table ..................................................................................... 23

Discussion ................................................................................................ 33

References ................................................................................................ 38

Abstracts (Korean) .................................................................................... 50

5

Introduction

Tooth development is dependent on both morphogenesis and

differentiation of related cells highly regulated by epithelial-mesenchymal

interaction [1]. Tooth organogenesis is normally divided into bud, cap, and

bell stages, depending on the changes in epithelial-mesenchymal morphology,

position and function mediated by signaling centers such as the placode

during the bud stage and enamel knots during the last two stages [2]. Some

important signaling molecules include BMPs, FGFs, SHHs, WNTs, as well

as TGF-β and TNF proteins [2, 3]. Single molecules have been tested to

demonstrate their role in odontogenesis by investigating the defects resulting

from their absence [4-6]. However, further examinations are needed as the

molecules are never found alone at work in a developmental setting [7].

The underlying mechanism of odontogenesis is commonly investigated

through co-cultures of two or more cell types in vitro [8, 9], while recent

advances have shifted their focus towards inducing odontogenic responses

with conditioned medium (CM) [10]. Most references highlight the role of

CM derived from animal cell sources, such as mouse and rat [11-13], which

are the most commonly used model organisms for human odontogenesis.

Much research has gained insight into their cell properties and mechanisms;

however, rodents retain stem cells that continuously renew their teeth [14],

whereas humans have two sets of dentition, one of which is permanent [15].

CM from human cell cultures lacks both the foundation and affirmative data

6

for odontogenesis. Also, there is a need to evaluate a range of CM

concentrations to find the optimal level for odontogenic differentiation. This

study is the first report of odontogenic induction potential of concentrated

CM derived from the human dental epithelial cell line.

In this study, I examined the effect of CM from a stable epithelial cell line

established from Hertwig’s Epithelial Root Sheath/Epithelial Rests of

Malassez (HERS/ERM), which originate from human molars [16, 17], on

stem cells from human exfoliated deciduous teeth (SHED). Also, different

concentrations of CM were tested for comparison. The effects of human cell-

derived CM were analyzed by CCK-8 assay, Alizarin red S and DAPI staining,

real-time qPCR, and Western blotting. The results showed that CM increased

the number of mineralized nodules in differentiated SHED culture, and thus

enhanced the odontogenic potential of SHED in vitro in a concentration-

dependent manner.

7

Literature Review

I. Odontogenesis and related signaling factors

Odontogenesis, the mechanism of tooth development in organisms,

entails an organized series of queues that outlines different stages of tooth

morphogenesis. Tooth formation results from epithelial and mesenchymal

stem cells interacting with one another through extracellular signaling

molecules of transforming necrosis factors (TNFs), bone morphogenetic

proteins (BMPs), fibroblast growth factors (FGFs), wingless/integrated

(WNTs), and sonic hedgehog (SHH) families to generate an inductive

environment [3]. The signaling pathway and regulatory molecules involved

are known to be conserved in embryonic development, and the specific

epithelial-mesenchymal interactions are well identified for each step of tooth

organogenesis [18]. These communications are mediated by several signaling

centers during tooth development. For instance, the placode, formed by the

thickening of ectoderm, signals the underlying neural-crest mesenchyme to

condense around the epithelial bud [2], and the primary and secondary enamel

knots regulate morphogenesis of the crown during bud and cap stage [19].

Tooth crown is finalized in bell stage when the epithelial and mesenchymal

cells differentiate into ameloblasts and odontoblasts at the interface and

deposit hard tissues, enamel and dentin matrices, respectively [2].

8

i. Conditioned medium in vitro and in vivo

Odontogenesis has been explored in vitro and in vivo through co-cultures

of epithelium and mesenchymal cells [8], in vivo therapy [6], induction by

conditioned medium (CM) [11, 13, 20], and so on. However, considering the

difficulty in obtaining and maintaining stem cells from human sources as well

as transplant rejection or engraftment issues and risk of developing cancer in

clinic applications, CM seems to be the most viable option.

CM has been studied in cell cultures with diverse cells or media types and

culture or disease conditions for a wide range of applications [10]. Few

disease conditions include injuries in liver [21, 22], lung [23, 24], spinal cord

[25] and brain [26, 27], in which CM improved the circumstances. CM

composition also varied between cell monolayer and spheroids [28]; it has

been reported that spheroid conditions produced higher concentrations of

growth factors and cytokines in CM than monolayer conditions [29]. Hypoxic

conditions also enriched paracrine factors in CM, subsequently enhancing its

effect on proliferation and tubular formation of cells [30]. Other variations in

culture duration, basal medium and supplements also affected CM

configuration [10]. Due to such a vast range of possibilities and deviations, it

is open to accommodate a large variety of in vitro and in vivo experiments in

numerous fields of study.

In vitro studies have used the paracrine factors within CM to promote

growth [31], repair [30] or differentiation [8, 32] of cells. Cellular factors,

such as cytokines and signaling molecules, have been investigated to enhance

9

cell performance [5, 33]. Previous studies have determined single factors that

influenced cell fate in vitro [34, 35], but their mode of action when combined

is uncertain. Thus, others have analyzed the composition of CM to help

narrow down the combination of elements at work [25, 36-38]. Tooth

formation is also governed by a network of growth and transcription factors

that shift the odontogenic potential back and forth between epithelium and

mesenchyme at E12.5 [3]. Likewise, tooth germ cell-derived CM, which is

thought to contain regulators from both epithelium and the surrounding

mesenchyme, was used to differentiate adult stem cells into odontoblast-like

cells in vitro [13]. In other words, CM is able to reproduce the temporospatial

effect of an odontogenic microenvironment of in vivo state required for

cellular transformation [13, 39]. However, the regulation of these molecules

are complex and the specific roles for each are difficult to assign because most

have multiple roles, some are redundant, and together they have synergistic

effects [7].

CM also received attention as a novel therapeutic method to overcome the

limitations of stem cell therapy [36]. This cell-free system has been

functionally verified in in vivo angiogenesis [36, 40], liver regeneration [22],

and odontogenesis [12] to name a few. Another study inserted preameloblast-

derived CM in a hollow root canal space in mice with human pulp cells, which

successfully restored pulp tissue [11]. Moreover, a study comparing porcine

and human tooth germ cell-derived CM showed that both had similar effects

on the odontogenic induction of human dental pulp stem cells in vitro and in

10

vivo as indicated by morphological and genetic changes [20], raising hopes

for practical application of xenogeneic CM. Additionally, CM from cell lines

have been used as a tool for discovery of new biomarkers for diseases, and

analyses of various types of CM revealed cell-specific secretome, including

signal peptides, growth factors, enzymes and other soluble factors [41], which

may help identify specific factors for enhancement of clinical effect. Also,

unlike osteogenesis and mineralization, odontogenic-specific markers are not

well known, and thus CM may provide a list of potential biomarkers explicit

in odontogenesis.

ii. Conditioned medium contains extracellular vesicles

Like the odontogenic environment of tooth formation in vivo, the key

premise of CM is that it provides a microenvironment that is similar to the

actual odontogenic conditions, allowing cells within it to be reprogrammed.

For instance, tooth germ cell-derived CM contains biological factors such as

TGF-β and BMP molecules [7, 20] that play a role in cytodifferentiation and

directing cell fate [42, 43]. CM is also known to contain exosomes, a type of

extracellular vesicles gaining attention in recent years. Although the

mechanism of cellular communication in tooth development remains obscure,

molecules of sizes larger than 100 nm seem to be involved in the polarization

or differentiation of cells [44]. Exosomes, also within this boundary, were

able to induce amelogenesis or dentinogenesis depending on whether

mesenchymal exosomes were used on epithelium or vice versa. [45].

11

Exosomes in osteogenic conditions also increased mineralization nodules

and corresponding matrix proteins, and the effect was reported to be dose-

dependent [45-47]. Moreover, exosome treatment amplified DPP and DMP1

expression at the junction of dentin and soft tissue of exosome-treated

samples, indicating augmented matrix mineralization [46]. Similarly, co-

culture of two cell types with a permeable barrier in between allowed

transmission of cytokines, resulting in mineralized nodules [48]. Moreover,

Shapiro et al. (2015) claims that exosomes are related to matrix vesicles [49],

the initiating point of mineralization in bone and other mineralizing tissues

[50-53], despite the fact that the former is a non-adherent extracellular vesicle,

while the latter is always found attached to the extracellular matrix

components [49, 54]. They argued that the size of matrix vesicles, from 0.02

to 2.0 μm [55], were within range of the diameter of exosomes varying from

0.04 to 1.0 μm [56], and the structure and formation of matrix vesicles

appeared similar to the budding of exosomes [57, 58]. If matrix vesicles are

indeed a type of exosomes, the study of CM may contribute greatly to

unveiling the mechanisms behind osteo- and odontogenic mineralization.

iii. Limitations of conditioned medium

Unfortunately, CM also needs to overcome a couple of limitations for

clinic use. First, the media used for CM collection contains elements that are

not suitable for humans such as HEPES; and secondly, the concentration of

secretome from cell culture is too low to have a significant effect on humans

12

[29]. Also, the specific content of CM must be reviewed for each type or batch

and method of CM collection standardized before clinical use in the future for

consistency and safety issues. Nonetheless, clinic efforts have been made

recently to treat multiple sclerosis [59], hair loss [60], and aging of skin [61],

all of which presented a positive outlook on the potential uses of CM. Further

research is needed to test more cases, but overall CM appears to be a durable

solution in the long term.

II. Mineralization, apoptosis and terminal differentiation

In organisms, apoptosis is known to play a significant role in embryonic

development as well as in tooth organogenesis [62, 63]. The shaping of the

tooth, silencing of signaling centers, and reduction of cell numbers are

primary examples of dental apoptosis [63]. For instance, as the dentin matrix

begins to occupy more space after secondary dentin deposition, the number

of odontoblasts are reduced by half through apoptosis [64]. Thus, a defect in

apoptosis may affect the overall dental development, influencing the number

or the size of teeth [65, 66].

A recent study reported the effect of calcium and mineralization of dental

pulp cells on apoptosis [67]. Although the reasons for cell death is unclear,

the higher calcium ion concentration of 5.4 and 9.0 mM increased apoptotic

and necrotic markers [67]. Moreover, proinflammatory cytokines also induce

odontogenic differentiation of dental pulp stem cells [68, 69]. This may be

closely related to apoptotic bodies’ potential role as nucleation sites for

13

calcification as it is with matrix vesicles [70-72]. Terminal differentiation of

growth plate chondrocytes is also associated with apoptosis [73, 74], and a

factor regulating differentiation and apoptosis, activating transcription factor

2 (ATF-2), was statistically more expressed in terminally differentiated

odontoblasts than in pulpal fibroblasts [75]. Still, not much is known about

the correlation between mineralization, cell death and terminal differentiation

in odontogenesis, and thus requires further investigation to make a statement.

14

Materials and Methods

Primary cell culture

To culture SHED, human deciduous teeth were obtained in Hank’s

balanced salt solution (HBSS; Welgene, Daegu, Korea) supplemented with

3% antibiotics-antimycotics (Gibco, Carlsbad, CA, USA) from patients who

gave informed consent. Dental pulp tissues were extracted with fine forceps

and digested in 1 mg/ml of Collagenase Type I (Gibco) and 2.4 mg/ ml of

Dispase (Gibco) at 37°C. After one hour of incubation, the

Collagenase/Dispase enzyme solution was inactivated by Dulbecco’s

modified Eagle’s medium (DMEM; Welgene) supplemented with 10% fetal

bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% antibiotics-

antimycotics (Gibco). Single cell suspensions were seeded and maintained in

minimum essential medium alpha (αMEM; Hyclone) supplemented with 10%

FBS and 1% antibiotics-antimycotics. The medium was changed every two

days. Cells were sub-cultured at 70% confluence.

HERS/ERM cells were isolated and cultured as described by Nam et al.

[1]. Briefly, human third molars were obtained in HBSS (Welgene)

supplemented with 3% antibiotics-antimycotics as explained above.

Periodontal ligament tissues, gently separated from the surface of the root,

were incubated in Collagenase/Dispase enzyme solution at 37°C. After one

hour of incubation, the enzyme was inactivated by DMEM supplemented

with 10% FBS and 1% antibiotics-antimycotics. Single cell suspensions were

15

seeded and maintained in αMEM supplemented with 10% FBS and 1%

antibiotics-antimycotics. The medium was changed every two days. When

colonies were observed, the mesenchymal cells were isolated by

trypsinization with 0.05% trypsin-EDTA (Gibco) and the remaining cells in

the culture plates were washed once with Dulbecco’s Phosphate-Buffered

Saline (DPBS; Welgene) and maintained in serum-free keratinocyte growth

medium-2 (KGM-2; Lonza Rockland, Rockland, ME, USA). Cells were sub-

cultured at 70% confluence.

Preparation and application of CM

To prepare human epithelial CM, HERS-SV40/hTERT cell line was

counted and cultured in vitro for 24 hours in serum-free growth medium,

KGM-2. After washing with DPBS, the cells were cultured in fresh KGM-2

for 48 hours. The conditioned medium was collected, centrifuged at 2000 x g

for 10 minutes at 4°C, then filtered by 0.22 μm filter membrane (Millipore,

Billerica, MA, USA) to eliminate cell debris before aliquoting and freezing

at -80°C. Frozen CM was lyophilized using a freeze-dryer (IlShin BioBase,

Dongducheon, South Korea) at -80°C and 5 mTorr for a week (Figure 1A).

The lyophilized powder was dissolved in a known volume of distilled water

to concentrate the CM by a factor of 16.

In cell culture, the culture media was switched to either non-induction

control media (αMEM supplemented with 5% FBS) or induction media

(αMEM supplemented with 5% FBS, 50 μg/ml ascorbic acid, 10 mM β-

16

glycerophosphate, and 0.1 μM dexamethasone) supplemented with or without

HERS/ERM CM or freeze-dried CM (Figure 1B) when SHED reached

confluence. HERS/ERM CM was added to constitute 10% of the medium,

while HERS/ERM freeze-dried CM was supplemented at 4X and 8X

concentrations, which are equivalent to 40% and 80% constitution,

respectively.

Cell Counting Kit-8 (CCK-8) cytotoxicity assay

Cells were counted and dispensed (5000 cells/100 μl/well) in a 96-well

plate. The plate was incubated for 24 hours in a humidified incubator (5%

CO2, 37°C), and CCK-8 solution was added to each well of the plate. After

two hours of incubation, the absorbance of the wells was measured at 450 nm

using a microplate reader (BMG LABTECH, Ortenberg, Germany). Media

was changed every day, and the absorbance was measured at the same time

each day for six days.

Alizarin red S staining

Primary SHED was cultured to confluence and cultured for 8, 12, 16, and

20 days in differentiation induction media, αMEM supplemented with 5%

FBS, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 0.1 μM

dexamethasone with varying CM and freeze-dried CM concentrations. The

medium was changed every other day. To visualize the accumulation of

mineralized nodules, the differentiated SHED were stained with 2% Alizarin

17

red S solution (Sigma-Aldrich, St. Louis, MO, USA). The cells were washed

with DPBS and fixed with 4% paraformaldehyde (T&I Biotechnology, Seoul,

Korea) at room temperature. After washing with DPBS, the cells were stained

with 2% Alizarin red S solution (Sigma-Aldrich). The remaining Alizarin

solution was washed with distilled water.

DAPI staining

Primary SHED was cultured to confluence in 12-well plates (SPL,

Pocheon, Korea) and cultured for 10, 12, 14, 16, or 18 days in differentiation

induction media. On the day of DAPI staining, cells were fixed with 4%

paraformaldehyde at room temperature. After washing with DPBS, DAPI

(Sigma) diluted (1:1000) in DPBS was added in the dark and incubated at

room temperature for 10 minutes. The remaining DAPI was washed with

DPBS and pictures were taken using an inverted microscope (Nikon, Melville,

NY, USA). All immunofluorescence staining were conducted more than three

times.

Reverse transcription-polymerase chain reaction (RT-PCR)

Cell pellets in RNAlater (Ambion, Austin, TX, USA) were washed with

DPBS supplemented with 2% FBS. Total RNA isolation was performed using

the RNeasy mini kit (Qiagen, Hilden, Germany). An additional DNase I

treatment from the RNase-free DNase set (Qiagen) was done for removal of

genomic DNA contamination. Total RNA (1 μg) was reverse transcribed

18

using the amfiRivert cDNA synthesis Platinum Master Mix (GenDEPOT,

Barker, TX, USA). The following RT conditions were used: annealing at 25°C

for 5 min, followed by extension at 42°C for 60 min, and heat inactivation of

the enzyme at 70°C for 15 min. The RT products were stored at -20°C until

quantitative analysis.

Real-time quantitative PCR (RT-qPCR)

The cDNA reverse transcribed from SHED were amplified using

Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) and specific primers

for human BSP, Runx2, ON, OC, DMP1, MEPE, and GAPDH listed in Table

1. Real-time PCR was performed by CFX CONect Real-Time PCR Detection

System (Bio-Rad, Hercules, CA, USA). The cycle threshold (CT) values of

each gene were standardized to those of the housekeeping gene,

glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following PCR

conditions were used: initializing at 95°C for 30 sec, denaturing at 95°C for

10 sec, annealing at 60°C for 30 sec, followed by elongation at 72°C for 33

sec, and heat inactivation of the enzyme at 70°C for 15 min.

Western blot analysis

Primary SHED was differentiated for 16 and 20 days with CM and freeze-

dried CM supplements, then collected in 1.5 ml tubes and pelleted by

centrifugation at 13,200 rpm, 4°C, for 3 minutes. Pellets were re-suspended

in RIPA lysis buffer (RKMB-030-0050, Rockland Immunochemicals,

19

Limerick, PA, USA) supplemented with Halt Protease and Phosphatase

Inhibitor Cocktail (78440, ThermoFisher Scientific, Waltham, MA, 1:100)

and incubated on ice. Proteins were separated by 10% sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to

immunoblot PVDF membrane (Bio-Rad). Membranes were blocked with 5%

non-fat dry milk (Bio-Rad) in TBS-T [20 mM Tris base, 137 mM NaCl, 0.1%

Tween 20 (pH 7.6)], and incubated overnight at 4°C with primary antibody

diluted in TBS-T buffer (1:1000). Monoclonal antibody against DSPP (sc-

73632, Santa Cruz), binding to the DPP domain CSRGDASYNSDESKDNG

[76], was purchased from Santa Cruz Biotechnology (Santa Cruz

Biotechnology, CA, USA). After washing with TBS-T buffer, membranes

were incubated with goat anti-mouse IgG secondary antibodies conjugated to

horseradish peroxidase (BR170-6516, Bio-Rad). Labeled protein bands were

detected using a bio-image analyzer (Bio-Rad).

Statistical analysis

The data were presented as means ± standard deviations of three replicates

for each treatment. Data analysis was performed using analysis of variance

(ANOVA) and post hoc LSD t-test and Duncan for pair-wise comparisons.

The differences were considered to be significant when p < 0.05. All statistical

analyses were conducted using SAS software (version 9.4).

20

Results

The viability of SHED in HERS/ERM freeze-dried CM treatment

A toxicological test was performed for a week to examine the effects of

HERS/ERM freeze-dried CM on the viability of SHED at different

concentrations in vitro. The CCK-8 assay indicated that cell count was not

greatly altered from the control up to 8X concentration, but the absorbance

measured dramatically dropped at 16X and 32X concentrations after just one

day of incubation. At 8X concentration, the absorbance measurement was

visibly lower than the control, but cells still had no difficulty reaching

confluence by the end of the week. The 1X, 2X and 4X freeze-dried CM

treatments seemed to follow the same trend as the control (Figure 2).

HERS/ERM CM induces odontogenic differentiation of SHED in vitro

After 8 and 12 days of CM treatment, differentiated SHED was analyzed

by RT-qPCR to determine whether CM enhanced odontogenesis and

mineralization (Figure 3). The mineralization-related BSP expression was

significantly increased in freeze-dried CM treatments on both day 8 and 12,

while DMP1 was significantly enhanced in 4X CM treatment on both days

compared to the controls and in 8X CM treatment on day 12. MEPE

expression was also significantly increased in freeze-dried CM groups on day

8 and 12 when compared to the freeze-dried basal media controls. On the

other hand, early mineralization markers like RUNX2 showed a decreasing

21

trend in all treatment on day 12 compared to the non-induction control. Other

early markers, such as ON and OC, also showed a similar trend as RUNX2.

Treatment groups had relatively higher gene expression levels on day 8 but

declined on day 12. The relative ON expression in basal media control groups

was higher than CM treatment on both days, but the 4X control showed

significantly less OC expression on both days.

Alizarin red S staining was also performed to compare the calcification

nodule formation of CM-treated groups to the non-induction control. Staining

results exhibited no staining of nodules on day 8. The first signs of

mineralized nodule formation were observed on day 12 in CM-treated groups.

Odontogenic induction without CM exhibited mineralized nodules on day 16

and increased onwards, while CM-treated groups showed a notable increase

in nodule formation from day 12 to day 20 (Figure 4A). All induction groups

excluding basal media controls were stained thoroughly on day 20, but in

earlier days, the difference between the amounts of nodule formation was

prominent among these treatment groups. In contrast, the non-induction

group and basal media control groups were not stained for the duration of 20

days. At 200X magnification, SHED had larger Alizarin stained portions as

the concentration of CM treatment was increased (Figure 4B). The addition

of 1X CM also increased the number of nodule formation compared to the

induction group without CM, but not more than the freeze-dried CM

treatments. A similar pattern was also observed in unstained morphology of

differentiated SHED, where the freeze-dried CM-treated cells had

22

significantly more visible mineralized aggregates compared to the non-

induction and basal media controls. The aggregates also appeared to be more

densely formed in higher concentrations of CM compared to the 1X CM

group (Figure 6A).

Furthermore, Western blot was run to verify that mineralization in vitro

occurred as a result of odontogenic differentiation of SHED. The

immunoblots demonstrated that the relative amount of DPP proteins was

increased in freeze-dried CM treatment groups on day 16 compared to the

non-induction and induction controls. On day 20, the DPP expression

increased in all treatments compared to the non-induction control (Figure 5).

Terminal differentiation of SHED when treated with HERS/ERM freeze-

dried CM

To test the viability of SHED under the influence of freeze-dried CM over

a longer period, DAPI staining experiment was conducted on day 18. The

non-induction and induction controls showed consistent morphology of

SHED nuclei throughout 18 days. On the other hand, 4X and 8X CM-treated

nuclei began to alter and showed similar signs to DNA fragmentation on day

18. This data conforms to the expression of β-actin in western blotting, in

which 4X and 8X CM-treated cells exhibited less amount of β-actin, despite

loading the equal quantity of proteins for all treatments. By day 16 and 20,

both freeze-dried CM-treated groups had significantly less expression of β-

actin proteins compared to the controls (Figure 5).

23

Table 1. Sequences of primers used for RT-PCR

Class Gene Size

(bp)

Forward Sequence

(5’ 3’)

Reverse Sequence

(5’ → 3’)

Odontoblast

Markers

hBSP 100 AAG GCT ACG ATG GCT ATG ATG GT AAT GGT AGC CGG ATG CAA AG

hRUNX2 149 CCC AGT ATG AGA GTA GGT GTC C GGG TAA GAC TGG TCA TAG GAC C

hON 100 TAC ATC GGG CCT TGC AAA TAC GGG TGA CCA GGA CGT TCT TG

hOC 235 ATC CTT TGG GGT TTG GCC TAC GCC AAT AGG GCG AGG AGT G

hDMP1 265 ACA GGC AAA TGA AGA CCC TTC ACT GGC TTG TAT GG

hMEPE 116 CAA GAA GCC AGG TAT TCT GAA GG TGT GGT TGA AAT GTT GGT GCT

GAPDH 153 CAT CAC TGC CAC CCA GAA GAC TG ATG CCA GTG AGC TTC CCG TTC AG

24

A

B

Figure 1. Procedure outline for establishing the HERS/ERM freeze-dried conditioned medium (CM) used in the following experiments.

(A) The HERS/ERM cell line was seeded 60 x 104 cells per 90 mm dish in serum-free growth medium and the CM was collected after

48 hours of incubation. The CM was centrifuged at 2000 x g for 10 minutes, filtered with a 0.22-μm membrane filter, and frozen at

-80°C before lyophilization. By adding a known volume of distilled water to the freeze-dried CM powder, the CM was concentrated

by a factor of 16. (B) The SHED was cultured in induction medium supplemented (c) with or (b) without CM, or (e) with freeze-dried

CM obtained from the HERS/ERM cells, and compared to the SHED cultured in (a) non-induction medium, or in (d) induction

medium supplemented with freeze-dried basal medium (BM).

25

Figure 2. Toxicological test of freeze-dried conditioned medium (CM) on the SHED in vitro. The SHED was treated with growth

medium supplemented with the HERS/ERM CM at six different concentrations, 1X, 2X, 4X, 8X, 16X, and 32X, and compared to the

control; the value of 1X represents the addition of 10% (v/v) CM. The SHED was seeded at Day 0, and the first absorbance was

measured at Day 1, the day on which the CM were added. Cell viability was assessed every 24 hours for a week by CCK-8 assay. The

concentration of 16X and 32X led to immediate cell death, while all other treatment groups eventually reached cell confluence by the

last day. The 8X group exhibited a slower growth rate compared to lower concentration groups, but reached confluence by the end of

the week. Data are mean ± SD of three replicates.

26

Figure 3. Relative gene expression of differentiated SHED in vitro. The SHED was treated with induction medium supplemented with

different concentrations of HERS/ERM conditioned medium (CM) for 8 and 12 days, where the value of 1X represents the addition

of CM at 10% (v/v) of the total volume. The relative mRNA expression levels of BSP, DMP1, MEPE, RUNX2, ON and OC were

27

measured by quantitative RT-PCR analysis and the data were normalized by GAPDH levels. The HERS/ERM freeze-dried CM-treated

groups showed a significant increase in BSP, DMP1, and MEPE expression compared to the controls, while early mineralization

markers, RUNX2, ON and OC, showed significant decreases on day 12. Data are mean ± SD of three replicates. Bars labeled with

different letters or numbers are significantly different from the non-induction control (1,2,3p < 0.05 and a,b,cp < 0.05). BSP: bone

sialoprotein; DMP1: dentin matrix acidic phosphoprotein 1; MEPE: matrix extracellular phosphoglycoprotein; RUNX2: Runt-related

transcription factor 2; ON: osteonectin; OC: osteocalcin.

28

A

Figure 4. Mineralized nodule formation of differentiated SHED in vitro. The SHED was cultured in induction medium supplemented

with different concentrations of HERS/ERM conditioned medium (CM), where the value of 1X represents the addition of 10% (v/v)

CM. The label indicates the presence of induction and/or CM. The calcium nodule formation was observed through (A) Alizarin red

S stained cells after 8, 12, 16 and 20 days of culture. Differentiated SHED produced more mineralized nodules when supplemented

with CM, and mineralization density increased as CM concentration was increased. All experiments were conducted in three replicates.

29

B

Figure 4. (B) Alizarin red S stained cells after 12, 16 and 20 days of culture, photographed at 200X magnification. Mineralized nodules

were observed 12 days after the start of odontogenic induction, and accumulated until all differentiated groups reached mineralization

equilibrium after 20 days. None of the controls were Alizarin red S stained. All experiments were conducted in three replicates.

30

Figure 5. Western blot analysis of differentiated SHED in vitro. The SHED was cultured in induction medium supplemented with

different concentrations of HERS/ERM conditioned medium (CM) for 16 and 20 days, where the value of 1X represents the addition

of CM at 10% (v/v) of the total volume. The label indicates the presence of induction and/or CM. The expression levels of DPP and

β-actin proteins were analyzed by western blotting. DPP was more strongly expressed in CM-treated groups compared to the non-

induction and induction controls. The disappearance of DSPP bands (solid arrows) corresponded with the appearance and the thickness

of DPP bands (dotted arrows). The β-actin protein bands were thinner in freeze-dried CM-treated groups compared to the basal media

control bands. Lane 1: non-induced SHED; lane 2: odontogenic induced SHED; lane 3: odontogenic induced SHED treated with 4X

freeze-dried basal media (BM); lane 4: odontogenic induced SHED treated with 8X freeze-dried BM; lane 5: odontogenic induced

SHED treated with 4X freeze-dried CM; lane 6: odontogenic induced SHED treated with 8X freeze-dried CM.

31

A

Figure 6. Cell morphology of differentiated SHED in vitro. The SHED was cultured in differentiation medium supplemented with

different concentrations of HERS/ERM conditioned medium (CM), where the value of 1X represents the addition of CM at 10% (v/v)

of the total volume. The label indicates the presence of induction and/or CM. (A) Cell morphology was observed 20 days after

odontogenic induction. Cells treated with freeze-dried CM was highly mineralized after 20 days of odontogenic induction, compared

to the basal media controls which showed no mineral nodule formation.

32

B

Figure 6. (B) Cell nuclei were stained by DAPI after 18 days of induction and photographed at 600X magnification to observe nuclear

alterations. Microscopic images showed signs of DNA fragmentation in freeze-dried CM-treated cells, while the non-induction and

induction control groups maintained smooth, oval-shaped nuclei. The pictures are representative of three independent experiments.

33

Discussion

Tooth development is a complex, regulated series of reciprocal

communication between the epithelium and mesenchyme, which eventually

differentiate into ameloblasts and odontoblasts, respectively, to form hard-

tissue [2]. To reproduce cellular interaction, stem cells have been used for in

vitro co-culture systems [8] or bioengineering for tooth regeneration [77].

However, the invasiveness of stem cells has made the transition to bedside

difficult, as well as other issues including the expenses and maintenance of

cell culture. CM, on the contrary, are proven to have regenerative effects and

are easily obtained from cell cultures.

CM has been established from various cell types and culture conditions,

and have been utilized in the induction of cell differentiation in vitro [8, 78]

and in vivo [11, 79, 80]. It is said to contain soluble factors [81-83] that create

an odontogenic microenvironment favorable for cell differentiation [13].

However, the effect of cytokines and other paracrine factors on the regulation

of cell function is complex, and thus, the results are not always dose-

dependent. For instance, one study found that the concentration of CM

supplements did not correlate with cell proliferation [84]. Others found that

varying concentrations of CM had no effect on the cell behavior [40, 85].

Interestingly, the CM in our study had concentration-dependent

enhancing effects on the odontogenic capacity of SHED. This could be due

to our method of concentrating the CM by lyophilization. After collecting and

34

freezing the CM from HERS/ERM cell line, the frozen CM was freeze-dried,

and the resultant powder was dissolved in a known volume of distilled water

to concentrate the CM by a factor of 16. The rationale behind this was to

increase the concentration of CM supplement in differentiation medium

without constituting a larger proportion of the total volume. Thus, I was able

to add 40% and 80% of CM (v/v) to the differentiation medium by adding

only 25 and 50 μl of CM, respectively, per milliliter of odontogenic induction

medium.

In vitro differentiation of SHED with the supplementation of freeze-dried

CM was examined by mineralization-related gene expression levels, namely

BSP, DMP1, MEPE, RUNX2, ON, OC, and as well as the expression of DPP

proteins. Previous studies state that BSP is expressed in mature osteoblasts

[86, 87], and its appearance correlates to calcified nodule formation [88]. On

both day 8 and 12, BSP was significantly increased in higher concentrations

of CM (Figure 3), and Alizarin red S staining exhibited increased formation

of mineralized nodules in differentiated SHED with 4X and 8X freeze-dried

CM supplements on day 12 (Figure 4B). Similar to BSP, DMP1 is also

associated with the start of mineralization [89]. As expected, the mRNA

expression of DMP1 was enhanced significantly in 4X freeze-dried CM

treatment; but data was not significant in 8X freeze-dried CM treatment

(Figure 3A). This may be due to inconsistent effects of 8X freeze-dried CM

on SHED differentiation, as observed by the slight variations between

replicates in Alizarin red S staining on day 12 and 20 (Figure 4A). Also,

35

MEPE is reported to be present in mineralizing tissues as its cleaved form,

acidic-serine-aspartate-rich-MEPE-associated motif [90, 91]. Unlike BSP,

DMP1, and MEPE, the expression of RUNX2, ON, and OC was significantly

downregulated in CM treatments on day 12, which is consistent with the

claim that RUNX2 expression declines in more mature odontoblasts [92],

while ON and OC are inversely correlated with calcium deposition [93, 94].

Lastly, DPP proteins that are closely related to odontogenesis and

mineralization in vitro [95] exhibited thicker bands in differentiated SHED

compared to the non-induction control on immunoblot, and bands at higher

sizes around 131 kDa seemed to disappear in correspondence to the

appearance and the thickness of lower DPP bands at 97 kDa [96]. Because

the monoclonal DSPP (LFMb-21) antibody binds to the DPP domain

CSRGDASYNSDESKDNG, both cleaved DPP and the remaining uncleaved

DSPP form should be detected on Western blot. However, there is still no

accepted explanation for the multiple bands detected by DSPP (LFMb-21)

antibody. Additionally, DPP bands were expressed highly in all differentiated

SHED on day 20 (Figure 5). This result was also consistent with Alizarin red

staining, which displayed significant levels of calcification nodules on day 20

(Figure 4A).

To confirm that the increased odontogenic effect on SHED was due to the

conditioning of medium by epithelial cells for 48 hours, the unconditioned

basal medium was added to the induction medium as a control variable. The

basal medium was freeze-dried and used at 4X and 8X concentrations to

36

compare directly with the freeze-dried CM treatments. As a result, the lack of

significant differences in relative gene levels, mineralized nodule deposits,

and Western blot in the basal media controls verified that the enhancement of

odontogenic capacity was due to the HERS/ERM cell line conditioning of the

basal medium, and not its original components.

However, it appeared that long-term treatment of freeze-dried CM

resulted in cell death, and the nuclei appeared to disintegrate, as determined

by DAPI staining (Figure 6B) and the decreasing β-actin expression in

Western blotting (Figure 5B). This could be due to the accumulation of freeze-

dried CM toxicity in long-term cell cultures, which was also observed at

higher concentrations of freeze-dried CM according to the CCK-8 assay. Cell

growth and viability were not greatly affected up to 8X concentration but

resulted in immediate cell death at 16X and 32X concentrations on the first

day of absorbance measurement (Figure 2). However, freeze-dried basal

media did not result in cell death on day 18, indicating that concentrated levels

of basal media components were not the cause of cellular toxicity. Thus, it

could also be explained by the nature of the cytodifferentiation stage in tooth

development. At terminal differentiation of odontoblasts, the cell cycle stops,

and the cells elongate, polarize and secrete a dentin matrix [5]. Smooth, oval

shapes of the nuclei were maintained until day 14 in 4X freeze-dried CM

treatment and day 16 in 8X freeze-dried CM treatment (data not shown).

Therefore, cell death naturally occurs as cells are terminally differentiated by

CM, and without further cell proliferation due to confluence, only the secreted

37

pre-dentin matrix remains as asserted by the strong expression of DPP

proteins even when beta-actin was reduced.

Our results suggest that the odontogenic differentiation effects of CM

derived from HERS/ERM cell line on SHED were concentration-dependent

up to 8X concentration factor in vitro. In-depth studies are needed to reveal

the specific composition of the CM and its involvement in the odontogenic

differentiation mechanism of mesenchymal cells.

38

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국문초록

사람 치아 상피세포에 의한

치수 줄기세포의 상아모세포 분화 유도

채 근 영

서울대학교 대학원 치의과학과 분자유전학 전공

(지도교수 이 진)

치아 발생과 재생 과정은 상피와 외배엽성 간엽 줄기세포 간의 다양한

상호작용으로 이루어지며 치아를 구성하는 법랑질 및 상아질은 이러한

상피-간엽 상호작용을 통해 분화된 법랑모세포와 상아모세포로부터

형성된다. 현재까지의 치아재생 연구는 분리 및 유지가 용이한 치수

간엽줄기세포를 중심으로 이루어졌는데, 반면에, 치아 상피세포는 치아

발생이 끝나고 나면 대부분 소실되기 때문에 확보 및 활용이 어려워

관련 연구가 많이 이루어지지 못하였다. 최근 선행 연구를 통해 사람

치주조직으로부터 치아 상피세포인 Hertwig’s epithelial root

sheath/epithelial rests of Malassez (HERS/ERM)가 확보되었으며

HERS/ERM 세포주 또한 확립되었으나 아직까지 치아조직재생에 대한

HERS/ERM의 역할에 대해서는 충분한 검증이 이루어지지 않았다. 본

연구에서는 사람 유치 치수줄기세포(stem cells from exfoliated

deciduous teeth, SHED)에 HERS/ERM 세포주의 조건배지를 처리한 후

SHED의 상아모세포 분화 능력을 관찰함으로서 치아재생 과정의 상피-

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간엽 상호작용에서 HERS/ERM의 역할을 확인하고자 하였다. 사람 유치

치수에서 SHED를 일차 배양하였고, HERS/ERM 세포주를 48시간 동안

배양하여 얻은 조건배지를 SHED에 10, 40, 80%(v/v)로 처리하였다.

다만, 조건배지를 40%와 80%로 추가할 때에는 용량을 최소화시키기

위해 조건배지를 동결건조로 농축시킨 후 사용하였다. 조건배지를

처리한 SHED는 Alizarin red S staining, RT-qPCR, Western blot 및

DAPI 핵염색을 시행하여 상아모세포로의 분화 유무를 확인하였고,

세포배양 과정을 거치지 않은 조건배지의 basal media를 추가하여

대조군으로 사용하였다. Alizarin red S 염색 결과 조건배지의 농도가

증가함에 따라 12일에서 20일까지 칼슘 침착이 증가하는 것을 볼 수

있었다. 경조직의 특이 유전자인 BSP, DMP1과 MEPE의 발현도

조건배지의 농도가 증가함에 따라 8, 12일에 상승하였고, RUNX2, ON과

OC는 줄어드는 양상을 보였다. 반면에, 대조군으로서 조건배지의 basal

media만 추가한 경우 Alizarin red S 염색이 되지 않았으며 유전자 발현

양상도 분화에 따른 변화가 없었다. 상아모세포 표지단백질인 dentin

phosphoprotein 단백질 또한 16, 20일간 조건배지가 처리된 SHED에서

발현됨을 확인하였고, 대조군에서보다 더 많이 발현되는 것을

확인하였다. SHED에 조건배지를 처리하면서 장기간 배양하였을 때

대조군과는 달리 세포가 죽는 현상이 관찰되었고, DAPI 핵염색을 통해

관찰하였을 때 terminal differentiation과 연관있는 cell death와 같은

현상임이 추정된다. 이상의 연구결과를 종합하면 사람 HERS/ERM

세포주의 조건배지가 SHED의 상아모세포 분화를 유도하며 조건배지의

농도가 증가함에 따라 분화시기를 앞당기고 분화효율이 향상됨을 알 수

52

있었다. 따라서 HERS/ERM이 SHED의 상아모세포 분화에 미치는

영향은 조건배지를 통하여 상피에서 간엽간으로 정보가 전달됨으로써

이루어지는 것을 확인하였으나 분화유도물질의 발굴 및 동정과 같은

후속연구가 필요할 것이다.

……………………………………

주요어 : 사람 치주 조직 유래 상피세포, 사람 유치 치수 간엽줄기세포,

상피-간엽 상호작용, 조건배지, 치아 발생 및 재생, 상아모세포 분화

학 번 : 2017-24083