# Genetic Comparison of Stemness of Human Umbilical Cord and Dental Pulp

Authors: Chung-Min Kang; Hyunok Kim; Je Seon Song; Byung-Jai Choi; Seong-Oh Kim; Han-Sung Jung; Seok-Jun Moon; Hyung-Jun Choi
Journal: Stem Cells International
 (2016)

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## Abstract

This study focuses on gene expression patterns and functions in human umbilical cord (UC) and dental pulp (DP) containing mesenchymal stem cells (MSCs). DP tissues were collected from 25 permanent premolars. UC tissue samples were obtained from three newborns. Comparative gene profiles were obtained using cDNA microarray analysis and the expression of tooth development-associated and MSC-related genes was assessed by the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Genes related to cell proliferation, angiogenesis, and immune responses were expressed at higher levels in UC, whereas genes related to growth factor and receptor activity and signal transduction were more highly expressed in DP. Although UC and DP tissues exhibited similar expression of surface markers for MSCs, UC showed higher expression of CD29, CD34, CD44, CD73, CD105, CD146, and CD166. qRT-PCR analysis showed that CD146, CD166, and MYC were expressed 18.3, 8.24, and 1.63 times more highly in UC, whereas the expression of CD34 was 2.15 times higher in DP. Immunohistochemical staining revealed significant differences in the expression of genes (DSPP,DMP1, andCALB1) related to odontogenesis and angiogenesis in DP. DP and UC tissue showed similar gene expression, with the usual MSC markers, while they clearly diverged in their differentiation capacity.


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## Body

## 1. Introduction

Mesenchymal stem cells (MSCs) have attracted a great deal of interest because of their potential application in regenerative medicine and tissue engineering. MSCs are highly proliferative and adherent fibrotic cells that express characteristic cell surface markers and retain self-renewing capacity with the potential of differentiating into various tissues including bone, muscle, cartilage, fat, and nerve [1]. MSCs may be isolated readily from several tissues such as bone marrow, adipose tissue, and placenta [2]. Although MSCs derived from bone marrow are well characterized, the harvest of stem cells from the bone marrow is a highly invasive procedure with a considerable risk of donor site morbidity.Recently, potential alternative sources of MSCs and nonaggressive methods for the harvesting of stem cells have been investigated; in particular, the umbilical cord (UC) and dental pulp (DP) show great promise as source tissues because they contain a considerable number of cells with properties similar to those of MSCs [3, 4]. In recent years, UC-MSCs have shown an odontogenic differentiation potential to differentiate into odontoblast-like cells in an odontogenic microenvironment [5].UC tissue (Wharton’s jelly of human umbilical cord) is considered an ideal source of stem cells with characteristics similar to those of MSCs from bone marrow and adipose tissue, for example, fibroblast morphology, surface protein markers, and potential for differentiation into cells of therapeutic value [6, 7]. UC cells exhibit immunosuppressive capacity and high proliferation rates, which are useful in allogeneic environments [8, 9]. DP, which is thought to be derived from migratory neural crest cells during development, is a source of various populations of multipotent stem cells [10]. Similar to MSCs, dental pulp stem cells (DPSCs) are adherent clonogenic cells with varying capacities for differentiation along mesenchymal or nonmesenchymal lineages [11].The UC is a potentially valuable source of MSCs as the isolation process is noninvasive and causes no harm to the mother or infant and utilizes material that is usually discarded [12]. In addition, UC tissue is derived from the neonate and is therefore less mature than adult tissues. This lowers the risk of immune reactions during donor transplantation. DPSCs also represent a valuable source of MSCs, as the latter may be obtained with minimal pain and morbidity [13]. Therefore, MSCs derived from the UC and DP are more desirable for stem cell-based therapy than stem cells obtained from conventional sources such as bone marrow.Despite extensive knowledge of the properties of UC-MSCs and DPSCs, it is still not known whether their properties accurately reflect their true gene expression patterns and developmental potential in situ. The fate of stem cells is regulated by cell-intrinsic determinants and signals within a specialized microenvironment [14]. Therefore, investigation of the genes related to stemness in UC and DP is necessary in order to evaluate the potential value of these tissues as alternative sources of MSCs. In particular, their gene expression patterns must be analyzed to obtain insights into the differentiation capacity of the stem cells and to compare the biological functions of the stemness-related genes in terms of interactions with the microenvironment. In this study, we performed a DNA microarray-based differential gene expression analysis of UC and DP tissues with the aim of comparing the characteristics of MSCs from each type of tissue.


## 2. Materials and Methods

### 2.1. Tissue Samples

The experimental protocol was approved by the Institutional Review Board of Yonsei University Dental Hospital (#2-2012-0001) and Severance Hospital (#4-2012-0408). All the subjects or their guardians have provided written informed consent. Pulp samples were obtained from healthy permanent premolars (n=25; from 5 males and 6 females, aged 11–25 years) extracted for orthodontic reasons. The fresh umbilical cord tissues were obtained from three newborns in the Department of Obstetrics and Gynecology, Severance Hospital, Yonsei University. The extracted teeth and umbilical cords were frozen immediately and stored in liquid nitrogen. The pulp tissue was obtained using sterile tweezers and barbed broaches. The UC tissue was sliced at a thickness of 10–14 μm using a cryostat (CM3050S, Leica Biosystems, Newcastle Upon Tyne, UK). Subsequently, the DP and UC tissues were immediately submerged in Buffer RLT, which is a proprietary component of the RNeasy Fibrous Mini Kit® (Qiagen, Valencia, CA, USA).


### 2.2. RNA Extraction

We used similar study procedures applied by Song et al. [15], Lee et al. [16], and Kim et al. [17]. Total RNA was extracted from DP and UC tissues using the RNeasy Fibrous Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted RNA was eluted in 25 μL of sterile water. Prior to RNA extraction, tissues were homogenized using a Bullet Blender® Bead (Next Advanced, Averill Park, NY, USA). RNA concentrations were determined from absorbance values at a wavelength of 260 nm using a spectrophotometer (Nanodrop ND-1000®, Thermo Scientific, Waltham, MA, USA). RNA samples used in this study had 260/280 nm ratios equal to or greater than 1.8.


### 2.3. cDNA Microarray

Global gene expression analyses were performed using GeneChip® Human Gene 1.0 ST oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA). The average amount of RNA isolated from DP and UC tissues was 1μg. Total RNA was isolated using the RNeasy Fibrous Mini Kit columns as described by the manufacturer (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip® (Agilent Technologies, Amstelveen, Netherlands). RNA quantity was determined using the Nanodrop ND-1000. Each RNA sample was subjected to global gene expression analysis according to the manufacturer’s protocol (http://www.affymetrix.com/). Briefly, 300 ng of total RNA from each sample was converted to double-stranded cDNA. Using a random hexamer incorporating a T7 promoter, amplified RNA (cRNA) was generated from the double-stranded cDNA template via an in vitro transcription reaction and was purified using the Affymetrix sample cleanup module. cDNA was generated by random-primer reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented using the restriction endonucleases uracil-DNA glycosylase and human apurinic/apyrimidinic endonuclease. Next, the fragmented cDNA was end-labeled via a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the GeneChip Human Gene 1.0 ST array for 16 h at 45°C and 60 rpm as described in the GeneChip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). After hybridization, the chips were stained and washed in a GeneChip Fluidics Station 450 and scanned using a GeneChip Array scanner 3000 G7 (Affymetrix). The image data were extracted using Command Console software 1.1 (Affymetrix) and a raw file containing the expression intensity data was generated and used for the next step. This microarray data set was approved by the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/); the GEO accession numbers of the data set are GSE75642 (umbilical cord) and GSE75644 (dental pulp).


### 2.4. Gene Ontology Analysis

Expression data were generated using Expression Console software version 1.1 (Affymetrix). For normalization, the Robust Multiarray Average algorithm of the Expression Console software was used. In order to determine whether genes were differentially expressed in the three groups, a one-way ANOVA was performed on the Robust Multiarray Average expression values. A multiple testing correction was applied to thep values of the F-statistics to adjust the false discovery rate. Genes with adjusted F-statistic p values < 0.05 were extracted. Highly expressed genes that showed over 2-fold differences between the signal values in the control and each test group were selected for further investigation. In order to classify the coexpression gene group with a similar expression pattern, we performed hierarchical and K-mean clustering using MultiExperiment Viewer software 4.4 (http://www.tm4.org/, Dana-Farber Cancer Institute, Boston, MA, USA). The web-based tool, DAVID (the Database for Annotation, Visualization, and Integrated Discovery), was used for biological interpretation of differentially expressed genes. Subsequently, these genes were classified based on their function according to the KEGG Pathway database (https://david.ncifcrf.gov/home.jsp).


### 2.5. Quantitative Reverse Transcription Polymerase Chain Reaction

The single-stranded cDNA required in the PCR analysis step was produced using 500 ng of extracted total RNA as a template for reverse transcription (Superscript III Reverse Transcriptase and Random Primer, Invitrogen, Paisley, UK). The reverse transcription reaction was performed at 65°C for 5 minutes, followed by 25°C for 5 minutes, 50°C for 1 hour, and 70°C for 15 minutes to inactivate the reverse transcriptase. The synthesized cDNA was diluted 10 : 1 in distilled water and used as a template for qRT-PCR, which was performed using the ABI7300 RT-PCR system (Applied Biosystems, Warrington, UK). Samples of 25μL containing 1x Universal TaqMan Master Mix (4369016, Applied Biosystems), PCR primers at a concentration of 0.9 μM, and the diluted cDNA were prepared in triplicate. The amplification conditions were 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. TaqMan gene expression assay primers (Applied Biosystems) were used. The primers for each gene are listed in Table 1. ABI 7300 SDS 1.3.1 software (Applied Biosystems) was used to record the fluorescence intensity of the reporter and quencher dyes. Fluorescence intensity values were plotted against time and quantified as the cycle number. A precise quantification of the initial target was obtained by examining the amplification plots during the early log phase of product accumulation above background (the threshold cycle (Ct) number). Ct values were subsequently used to determine ΔCt values (ΔCt = Ct of the gene minus Ct of the 18S rRNA gene control), and differences in Ct values were used to quantify the relative amount of PCR product, expressed as the relative change by applying the equation 2-ΔCt. The specific primer assay ID and product sizes for each gene are listed in Table 1.Table 1
Quantitative RT-PCR primer lists.
Gene symbol
Gene description
Assay ID
Amplicon length
AMBN
Ameloblastin (enamel matrix protein)
Hs00212970_m1
61
ALCAM (CD166)
Activated leukocyte cell adhesion molecule
Hs00977641_m1
103
CALB1
Calbindin 1
Hs00191821_m1
90
CD34
CD34 molecule
Hs00990732_m1
91
DMP1
Dentin matrix acidic phosphoprotein 1
Hs01009391_g1
106
DSPP
Dentin sialophosphoprotein
Hs00171962_m1
67
MCAM (CD146)
Melanoma cell adhesion molecule
Hs00174838_m1
77
MYC
c-Myc
Hs00153408_m1
107
18S
18S rRNA
Hs03003631_g1
69


### 2.6. Immunohistochemical Staining

For IHC staining, tissues from permanent teeth and the UC were fixed in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) for 1 day. Permanent teeth were decalcified using 10% EDTA (pH 7.4; Thermo Fisher Scientific, Houston, TX, USA) for 8 weeks. The permanent teeth and UC tissues were embedded in paraffin and sectioned at a thickness of 3μm. Specimens were subjected to IHC staining with antibodies against DSPP (rabbit polyclonal, diluted 1 : 1500; sc-33586, Santa Cruz Biotechnology, Santa Cruz, CA, USA), DMP1 (rabbit polyclonal, diluted 1 : 100; Ab82351, Abcam), CALB1 (rabbit polyclonal, diluted 1 : 400; Ab25085, Abcam), and CD146 (MCAM, rabbit polyclonal, diluted 1 : 400; Ab75769, Abcam). Endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide. Sections were incubated in 5% bovine serum albumin to block nonspecific binding. The primary antibodies were diluted to obtain optimal staining and sections were incubated overnight. After incubation, the EnVision+ System-HRP Labeled Polymer Anti-Rabbit kit (K4003, Dako, Carpinteria, CA, USA; ready to use) was applied for 20 minutes. Color development was performed using labeled streptavidin biotin kits (Dako) according to the manufacturer’s instructions. The sections were counterstained with Gill’s hematoxylin (Sigma-Aldrich). Control sections were treated in the same manner but without primary antibodies.


## 2.1. Tissue Samples

The experimental protocol was approved by the Institutional Review Board of Yonsei University Dental Hospital (#2-2012-0001) and Severance Hospital (#4-2012-0408). All the subjects or their guardians have provided written informed consent. Pulp samples were obtained from healthy permanent premolars (n=25; from 5 males and 6 females, aged 11–25 years) extracted for orthodontic reasons. The fresh umbilical cord tissues were obtained from three newborns in the Department of Obstetrics and Gynecology, Severance Hospital, Yonsei University. The extracted teeth and umbilical cords were frozen immediately and stored in liquid nitrogen. The pulp tissue was obtained using sterile tweezers and barbed broaches. The UC tissue was sliced at a thickness of 10–14 μm using a cryostat (CM3050S, Leica Biosystems, Newcastle Upon Tyne, UK). Subsequently, the DP and UC tissues were immediately submerged in Buffer RLT, which is a proprietary component of the RNeasy Fibrous Mini Kit® (Qiagen, Valencia, CA, USA).


## 2.2. RNA Extraction

We used similar study procedures applied by Song et al. [15], Lee et al. [16], and Kim et al. [17]. Total RNA was extracted from DP and UC tissues using the RNeasy Fibrous Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted RNA was eluted in 25 μL of sterile water. Prior to RNA extraction, tissues were homogenized using a Bullet Blender® Bead (Next Advanced, Averill Park, NY, USA). RNA concentrations were determined from absorbance values at a wavelength of 260 nm using a spectrophotometer (Nanodrop ND-1000®, Thermo Scientific, Waltham, MA, USA). RNA samples used in this study had 260/280 nm ratios equal to or greater than 1.8.


## 2.3. cDNA Microarray

Global gene expression analyses were performed using GeneChip® Human Gene 1.0 ST oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA). The average amount of RNA isolated from DP and UC tissues was 1μg. Total RNA was isolated using the RNeasy Fibrous Mini Kit columns as described by the manufacturer (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip® (Agilent Technologies, Amstelveen, Netherlands). RNA quantity was determined using the Nanodrop ND-1000. Each RNA sample was subjected to global gene expression analysis according to the manufacturer’s protocol (http://www.affymetrix.com/). Briefly, 300 ng of total RNA from each sample was converted to double-stranded cDNA. Using a random hexamer incorporating a T7 promoter, amplified RNA (cRNA) was generated from the double-stranded cDNA template via an in vitro transcription reaction and was purified using the Affymetrix sample cleanup module. cDNA was generated by random-primer reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented using the restriction endonucleases uracil-DNA glycosylase and human apurinic/apyrimidinic endonuclease. Next, the fragmented cDNA was end-labeled via a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the GeneChip Human Gene 1.0 ST array for 16 h at 45°C and 60 rpm as described in the GeneChip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). After hybridization, the chips were stained and washed in a GeneChip Fluidics Station 450 and scanned using a GeneChip Array scanner 3000 G7 (Affymetrix). The image data were extracted using Command Console software 1.1 (Affymetrix) and a raw file containing the expression intensity data was generated and used for the next step. This microarray data set was approved by the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/); the GEO accession numbers of the data set are GSE75642 (umbilical cord) and GSE75644 (dental pulp).


## 2.4. Gene Ontology Analysis

Expression data were generated using Expression Console software version 1.1 (Affymetrix). For normalization, the Robust Multiarray Average algorithm of the Expression Console software was used. In order to determine whether genes were differentially expressed in the three groups, a one-way ANOVA was performed on the Robust Multiarray Average expression values. A multiple testing correction was applied to thep values of the F-statistics to adjust the false discovery rate. Genes with adjusted F-statistic p values < 0.05 were extracted. Highly expressed genes that showed over 2-fold differences between the signal values in the control and each test group were selected for further investigation. In order to classify the coexpression gene group with a similar expression pattern, we performed hierarchical and K-mean clustering using MultiExperiment Viewer software 4.4 (http://www.tm4.org/, Dana-Farber Cancer Institute, Boston, MA, USA). The web-based tool, DAVID (the Database for Annotation, Visualization, and Integrated Discovery), was used for biological interpretation of differentially expressed genes. Subsequently, these genes were classified based on their function according to the KEGG Pathway database (https://david.ncifcrf.gov/home.jsp).


## 2.5. Quantitative Reverse Transcription Polymerase Chain Reaction

The single-stranded cDNA required in the PCR analysis step was produced using 500 ng of extracted total RNA as a template for reverse transcription (Superscript III Reverse Transcriptase and Random Primer, Invitrogen, Paisley, UK). The reverse transcription reaction was performed at 65°C for 5 minutes, followed by 25°C for 5 minutes, 50°C for 1 hour, and 70°C for 15 minutes to inactivate the reverse transcriptase. The synthesized cDNA was diluted 10 : 1 in distilled water and used as a template for qRT-PCR, which was performed using the ABI7300 RT-PCR system (Applied Biosystems, Warrington, UK). Samples of 25μL containing 1x Universal TaqMan Master Mix (4369016, Applied Biosystems), PCR primers at a concentration of 0.9 μM, and the diluted cDNA were prepared in triplicate. The amplification conditions were 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. TaqMan gene expression assay primers (Applied Biosystems) were used. The primers for each gene are listed in Table 1. ABI 7300 SDS 1.3.1 software (Applied Biosystems) was used to record the fluorescence intensity of the reporter and quencher dyes. Fluorescence intensity values were plotted against time and quantified as the cycle number. A precise quantification of the initial target was obtained by examining the amplification plots during the early log phase of product accumulation above background (the threshold cycle (Ct) number). Ct values were subsequently used to determine ΔCt values (ΔCt = Ct of the gene minus Ct of the 18S rRNA gene control), and differences in Ct values were used to quantify the relative amount of PCR product, expressed as the relative change by applying the equation 2-ΔCt. The specific primer assay ID and product sizes for each gene are listed in Table 1.Table 1
Quantitative RT-PCR primer lists.
Gene symbol
Gene description
Assay ID
Amplicon length
AMBN
Ameloblastin (enamel matrix protein)
Hs00212970_m1
61
ALCAM (CD166)
Activated leukocyte cell adhesion molecule
Hs00977641_m1
103
CALB1
Calbindin 1
Hs00191821_m1
90
CD34
CD34 molecule
Hs00990732_m1
91
DMP1
Dentin matrix acidic phosphoprotein 1
Hs01009391_g1
106
DSPP
Dentin sialophosphoprotein
Hs00171962_m1
67
MCAM (CD146)
Melanoma cell adhesion molecule
Hs00174838_m1
77
MYC
c-Myc
Hs00153408_m1
107
18S
18S rRNA
Hs03003631_g1
69


## 2.6. Immunohistochemical Staining

For IHC staining, tissues from permanent teeth and the UC were fixed in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) for 1 day. Permanent teeth were decalcified using 10% EDTA (pH 7.4; Thermo Fisher Scientific, Houston, TX, USA) for 8 weeks. The permanent teeth and UC tissues were embedded in paraffin and sectioned at a thickness of 3μm. Specimens were subjected to IHC staining with antibodies against DSPP (rabbit polyclonal, diluted 1 : 1500; sc-33586, Santa Cruz Biotechnology, Santa Cruz, CA, USA), DMP1 (rabbit polyclonal, diluted 1 : 100; Ab82351, Abcam), CALB1 (rabbit polyclonal, diluted 1 : 400; Ab25085, Abcam), and CD146 (MCAM, rabbit polyclonal, diluted 1 : 400; Ab75769, Abcam). Endogenous peroxidase activity was quenched by the addition of 3% hydrogen peroxide. Sections were incubated in 5% bovine serum albumin to block nonspecific binding. The primary antibodies were diluted to obtain optimal staining and sections were incubated overnight. After incubation, the EnVision+ System-HRP Labeled Polymer Anti-Rabbit kit (K4003, Dako, Carpinteria, CA, USA; ready to use) was applied for 20 minutes. Color development was performed using labeled streptavidin biotin kits (Dako) according to the manufacturer’s instructions. The sections were counterstained with Gill’s hematoxylin (Sigma-Aldrich). Control sections were treated in the same manner but without primary antibodies.


## 3. Results

### 3.1. Gene Expression Profiles of UC and DP Tissues

Complementary DNA microarray technology was used to compare multiple gene expression profiles representative of DP and UC tissues. In order to investigate these differentially expressed genes further, data with a more stringent threshold of 3-fold differential expression were filtered to ensure biological significance. The results indicated that 1,957 out of 33,297 (5.88%) genes exhibited an absolute expression change at least 3-fold. The expression levels of 988 genes were 3-fold higher in the UC than in DP tissues, while the expression levels of 969 genes were at least 3-fold higher in DP than in UC tissues. The data were further filtered, and the genes are listed in Tables2 and 3 according to their biological functions.Table 2
Representative differentially expressed genes with higher expression levels in umbilical cord than in dental pulp tissue.
Functional category
Gene symbol
Biological process
Accession number
Absolute fold change
Embryonic development
DLK1
Embryonic skeletal system development
NM_003836
56.86
DKK1
Embryonic limb morphogenesis
NM_012242
44.51
SCEL
Epidermis development, embryo development
NM_144777
33.26
TBX18
Morphogenesis of embryonic epithelium
NM_001080508
15.66
HOXD10
Embryonic skeletal system morphogenesis
NM_002148
12.03
TBX20
Embryonic heart tube development
NM_001077653
11.71
HOXC10
Embryonic limb morphogenesis
NM_017409
8.70
HOXC6
Embryonic skeletal system development
NM_004503
7.22
HAND2
In utero embryonic development
NM_021973
6.54
HOXA6
Embryonic skeletal system morphogenesis
NM_024014
5.50
EDN1
In utero embryonic development
NM_001955
5.35
WNT4
Embryonic epithelial tube formation
NM_030761
4.31
GATA6
In utero embryonic development
NM_005257
4.17
TGFB1I1
Morphogenesis of embryonic epithelium
NM_001042454
4.13
TGFB3
In utero embryonic development
NM_003239
3.85
SOX5
In utero embryonic development
NM_152989
3.70
Developmental process
DES
Cytoskeleton organization
NM_001927
66.18
KRT6A
Ectoderm development
NM_005554
27.69
KRT5
Epidermis development
NM_000424
22.73
KRT13
Epidermis development, tongue morphogenesis
NM_153490
22.53
COL12A1
Skeletal system development, collagen fibril organization
NM_004370
17.87
DKK2
Multicellular organismal development
NM_014421
12.63
TAGLN
Muscle organ development
NM_001001522
11.91
KRT8
Cytoskeleton organization
NM_002273
11.04
KRT14
Epidermis development
NM_000526
9.01
OSTN
Ossification, multicellular organismal development
NM_198184
8.22
MCAM
Anatomical structure morphogenesis
NM_006500
7.22
IGF2BP3
Anatomical structure morphogenesis
NM_006547
7.10
ADAM19
Fertilization, muscle development, neurogenesis
NM_033274
6.41
MGP
Cartilage condensation, ossification
NM_001190839
5.70
DMD
Muscle organ development, skeletal muscle tissue development
NM_000109
5.22
PITX2
Multicellular organismal development
NM_153426
4.79
Physiological process
ACTG2
Muscle contraction
NM_001615
115.24
HBG1
Transport
NM_000559
101.65
PLN
Cellular calcium ion homeostasis, blood circulation
NM_002667
71.69
CNN1
Regulation of smooth muscle contraction
NM_001299
66.37
HBB
Regulation of blood pressure, oxygen transport
NM_000518
38.48
FGF10
Angiogenesis
NM_004465
31.24
COL8A1
Angiogenesis
NM_001850
12.02
AHSP
Hemoglobin metabolic process, hematopoiesis
NM_016633
7.18
FGF9
Angiogenesis, osteoblast differentiation
NM_002010
5.07
PDGFA
Angiogenesis, response to hypoxia
NM_002607
5.00
ELN
Respiratory gaseous exchange, blood circulation
NM_000501
4.75
EGF
Angiogenesis, positive regulation of cell proliferation
NM_001963
4.30
IL18
Angiogenesis, response to hypoxia
NM_001562
3.85
VEGFA
Angiogenesis, ovarian follicle development
NM_001025366
3.16
Signal transduction and regulation
PRLR
Cell surface receptor linked signaling pathway
NM_000949
90.61
RASSF3
Signal transduction
NM_178169
12.27
RERG
GTPase mediated signal transduction
NM_032918
11.98
STC2
Cell surface receptor linked signaling pathway
NM_003714
11.37
CD244
Signal transduction
NM_016382
8.83
ASPN
Negative regulation of transforming growth factor beta receptor signaling pathway
NM_017680
8.20
LPHN3
G-protein coupled receptor protein signaling pathway
NM_015236
7.72
SPSB1
Intracellular signaling pathway
NM_025106
6.87
ALCAM
Signal transduction, motor axon guidance
NM_001627
3.96
Cell regulation and proliferation
UPK1B
Epithelial cell differentiation
NM_006952
45.66
MYOCD
Cardiac cell differentiation
NM_001146312
40.32
EGFL6
Cell differentiation
NM_015507
11.94
PODN
Negative regulation of cell proliferation
NM_153703
9.96
FAS
Positive regulation of necrotic cell death
NM_000043
9.34
KRT4
Epithelial cell differentiation, negative regulation of epithelial cell proliferation
NM_002272
6.52
IGFBP7
Regulation of cell growth
NM_001553
6.01
HEMGN
Cell differentiation, regulation of osteoblast differentiation
NM_018437
5.70
CDH1
Trophectodermal cell differentiation
NM_004360
5.44
DPT
Cell adhesion, negative regulation of cell proliferation
NM_001937
5.13
Cytokine,  chemokine, and immune response
IL1RL1
Immune response
NM_016232
36.22
S100A8
Chemotaxis, inflammatory response
NM_002964
26.48
DPP4
Regulation of T cell mediated immunity
NM_001935
18.16
ITGB6
Inflammatory response
NM_000888
15.01
IGF2BP1
Regulation of cytokine biosynthetic process
NM_006546
9.59
LY96
Inflammatory response, cellular defense response
NM_015364
8.40
CCRL1
Chemotaxis, immune response
NM_178445
7.81
ANLN
Cytokinesis
NM_018685
7.60
CMKLR1
Chemotaxis, immune response
NM_001142343
7.41
CD97
Inflammatory response, immune response
NM_078481
6.96
CXCL1
Chemotaxis, inflammatory response, immune response
NM_001511
5.54
IL33
Positive regulation of macrophage activation
NM_033439
5.29
CXCR1
Chemotaxis, inflammatory response
NM_000634
3.48
CXCL6
Chemotaxis, inflammatory response
NM_002993
3.07Table 3
Representative differentially expressed genes with higher expression levels in dental pulp tissue than in umbilical cord.
Functional category
Gene symbol
Biological process
Accession number
Absolute fold change
Biomineral tissue development
PHEX
Bone and dentin mineralization
NM_000444
160.01
DMP1
Bone and dentin mineralization
NM_004407
99.20
CALB1
Hydroxyapatite formation
NM_004929
98.07
MMP20
Regulation of enamel mineralization, proteolysis
NM_004771
85.26
AMBN
Bone mineralization, odontogenesis of dentine-containing tooth
NM_016519
65.69
DSPP
Odontogenesis of dentine-containing tooth
NM_014208
41.26
ALPL
Biomineral tissue development
NM_000478
14.83
DLX1
Odontogenesis of dentine-containing tooth
NM_178120
9.55
LHX8
Odontogenesis of dentine-containing tooth
NM_001001933
7.56
CA2
Odontogenesis of dentine-containing tooth
NM_000067
7.07
RUNX2
Ossification, osteoblast differentiation
NM_001024630
6.53
LEF1
Odontogenesis of dentine-containing tooth
NM_016269
6.24
PAX9
Tooth development
NM_006194
5.79
MSX1
Odontogenesis, craniofacial development
NM_002448
4.04
WNT10A
Regulation of odontogenesis of dentine-containing tooth
NM_025216
3.87
Developmental process
DLX5
Osteoblast differentiation
NM_005221
30.01
DLX3
Multicellular organismal development
NM_005220
26.13
ADAM22
Proteolysis, central nervous system development
NM_021723
25.00
NES
Central nervous system development
NM_006617
15.67
BMP7
Ossification, organ morphogenesis
NM_001719
15.60
BMPR1B
Skeletal system development, cartilage condensation
NM_001203
13.53
MSX2
Skeletal system development, osteoblast differentiation
NM_002449
9.32
COL11A2
Skeletal system morphogenesis, cartilage development, palate development
NM_001163771
8.55
BMP5
Skeletal system development, ossification
NM_021073
6.08
TBX3
Blood vessel development, in utero embryonic development
NM_016569
5.93
MBP
Central nervous system development, myelination
NM_001025101
4.95
DLX6
Multicellular organismal development, nervous system development
NM_005222
4.12
NRCAM
Axonogenesis, central nervous system development
NM_001193582
4.09
SNAI1
Osteoblast differentiation, mesoderm formation
NM_005985
3.78
HLF
Multicellular organismal development, rhythmic process
NM_002126
3.23
BMP2
Development of bone and cartilage
NM_001200
2.11
Physiological process
TF
Controlling iron concentrations, erythropoiesis
NM_001063
153.98
SCN7A
Sodium ion transport, muscle contraction
NM_002976
82.36
APOD
Lipid metabolic process
NM_001647
41.22
PLAT
Response to hypoxia, blood coagulation
NM_000930
10.20
CD52
Elevation of cytosolic calcium ion concentration
NM_001803
4.29
CYGB
Response to oxidative stress, oxygen transport
NM_134268
3.98
MYOT
Muscle contraction
NM_006790
3.26
Signal transduction and regulation
WIF1
Wnt receptor signaling pathway
NM_007191
70.19
PTN
Transmembrane receptor protein signaling pathway
NM_002825
38.17
WNT5A
Oncogenesis, embryogenesis
NM_003392
13.68
NCAM1
Neurite outgrowth, synaptic plasticity
NM_181351
7.87
NCAM2
Neurite outgrowth, synaptic plasticity
NM_004540
7.38
RASGRF2
Induction of apoptosis by extracellular signals
NM_006909
6.88
CHN1
Signal transduction
NM_001822
6.56
MET
Cell surface receptor linked signaling pathway
NM_001127500
3.01
Cell regulation and proliferation
SCIN
Negative regulation of cell proliferation
NM_001112706
58.65
RELN
Cell morphogenesis involved in differentiation
NM_005045
49.12
NES
Structural organization of the cell
NM_006617
15.67
MEGF10
Phagocytosis
NM_032446
9.89
EPCAM
Positive regulation of cell proliferation
NM_002354
8.79
PDGFD
Positive regulation of cell division
NM_025208
5.43
CLU
Cell death, positive regulation of cell proliferation
NM_001831
5.19
VEGFC
Angiogenesis, positive regulation of neuroblast proliferation
NM_005429
3.74
MSI2
Stem cell development
NM_138962
3.55
DBC1
Cell cycle arrest, cell death
NM_014618
3.32
TGFB1
Cell growth, cell proliferation, cell differentiation and apoptosis
NM_000660
3.14
IGFBP6
Regulation of cell growth
NM_002178
3.07
Cytokine, chemokine, and immune response
IGJ
Immune response
NM_144646
22.65
IGHD
Immune response
BC021276
11.30
CXCL14
Chemotaxis, immune response, inhibiting angiogenesis
NM_004887
8.74
SELE
Leukocyte migration involved in inflammatory response
NM_000450
5.82
MX1
Induction of apoptosis, defense response, response to virus
NM_002462
5.24
IFI44
Response to virus
NM_006417
5.05
CX3CL1
Chemotaxis, defense response, immune response
NM_002996
3.60
CFI
Innate immune response
NM_000204
3.17


### 3.2. Gene Ontology Analysis

In the UC tissue, the expression levels of 158 genes were upregulated 10-fold or more in comparison with DP, whereas the expression levels of 118 genes were upregulated 10-fold in DP in comparison with UC. Genes related to cell proliferation and angiogenesis were expressed at higher levels in UC than in DP. Lipid metabolic process-related genes were highly expressed in DP. In comparison with DP, a large proportion of the UC genes were related to growth factor activity, structural molecular activity, DNA binding, and protein binding (Figure1).Figure 1
Main categories of genes expressed specifically in umbilical cord and dental pulp classified according to their biological (a) and molecular functions (b). Genes related to cell proliferation and angiogenesis were expressed at higher levels in the UC than in DP (p<0.05).
(a)
(b)


### 3.3. Stemness Characterization Using Surface Protein Markers

The comparative expression results for MSC surface protein markers are indicated in Figure3. UC tissue appeared to contain a population of cells that were more positive for MSC markers (includingCD29,CD34,CD44,CD73,CD105,CD146, andCD166) according to the minimal criteria of the International Society for Cell Therapy [18]. The comparative expression analysis of four induced pluripotent stem cell (iPSC) marker genes (i.e.,OCT4,SOX2,MYC, andKLF4) revealed that the expression level of these genes was the same in UC and DP. qRT-PCR analysis for eight important marker genes revealed four tooth-related genes (DSPP,AMBN,CALB1, andDMP1), three genes for MSCs (CD34,CD146, andCD166), and MYC related to iPSC stemness. DSPP, AMBN, CALB1, and DMP1 were expressed in DP tissue but they were not excluded in the comparative qPCR results because they were not detected in UC tissue. The expression levels of CD146, CD166, and MYC were 18.3, 8.24, and 1.63 times higher, respectively, in UC than in DP, and the expression level of CD34 was 2.15 times higher in DP than in UC (Figure 2).Figure 2
Relative gene expression of mesenchymal stem cell and induced pluripotent stem cell markers using cDNA microarray (a). The relative differences in the expression of stem cell markers between umbilical cord and dental pulp were analyzed using qRT-PCR (b). Data are presented as means ± standard deviation and expressed as the relative change by applying the equation2-ΔCt, where ΔCt = Ct of the gene minus Ct of the 18S rRNA.
(a)
(b)Figure 3
Immunohistochemical (IHC) staining of umbilical cord tissues. IHC staining for DSPP (a, e), DMP1 (b, f), CALB1 (c, g), and CD146 (d, h). CD146 is expressed on arteries of the umbilical cord, suggesting that the majority of stem cells arise from the microvasculature. (scale bars: (a)–(d) 4 mm, (e)–(h) 200μm).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


### 3.4. Immunohistochemical Staining

MCAM (CD146) was expressed abundantly as a perivascular stem cell marker in connective tissue, especially in the arteries of the UC. However, this protein showed a very low level of expression in DP tissue (Figure3). IHC staining showed that DSPP, DMP1, and CALB1 were expressed broadly in DP tissue (Figure 4), but barely expressed in the UC. DSPP was evident in pulpal tissue, the odontoblast layer, and primary and secondary dentin. While DMP was stained in the odontoblast layer and secondary dentin, CALB1 was expressed in pulpal tissue and the odontoblast layer. These findings were consistent with the microarray data.Figure 4
Immunohistochemical (IHC) staining of dental pulp tissue. IHC staining for DSPP (a, e). DSPP was noted in pulpal tissue, the odontoblast layer, and primary and secondary dentin. IHC staining for DMP1 (b, f) and CALB1 (c, g). CALB1 was especially expressed in pulpal tissue and the odontoblast layer. IHC staining for CD146 (d, h) (scale bars: (a)–(d) 4 mm, (e)–(h) 200μm).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.1. Gene Expression Profiles of UC and DP Tissues

Complementary DNA microarray technology was used to compare multiple gene expression profiles representative of DP and UC tissues. In order to investigate these differentially expressed genes further, data with a more stringent threshold of 3-fold differential expression were filtered to ensure biological significance. The results indicated that 1,957 out of 33,297 (5.88%) genes exhibited an absolute expression change at least 3-fold. The expression levels of 988 genes were 3-fold higher in the UC than in DP tissues, while the expression levels of 969 genes were at least 3-fold higher in DP than in UC tissues. The data were further filtered, and the genes are listed in Tables2 and 3 according to their biological functions.Table 2
Representative differentially expressed genes with higher expression levels in umbilical cord than in dental pulp tissue.
Functional category
Gene symbol
Biological process
Accession number
Absolute fold change
Embryonic development
DLK1
Embryonic skeletal system development
NM_003836
56.86
DKK1
Embryonic limb morphogenesis
NM_012242
44.51
SCEL
Epidermis development, embryo development
NM_144777
33.26
TBX18
Morphogenesis of embryonic epithelium
NM_001080508
15.66
HOXD10
Embryonic skeletal system morphogenesis
NM_002148
12.03
TBX20
Embryonic heart tube development
NM_001077653
11.71
HOXC10
Embryonic limb morphogenesis
NM_017409
8.70
HOXC6
Embryonic skeletal system development
NM_004503
7.22
HAND2
In utero embryonic development
NM_021973
6.54
HOXA6
Embryonic skeletal system morphogenesis
NM_024014
5.50
EDN1
In utero embryonic development
NM_001955
5.35
WNT4
Embryonic epithelial tube formation
NM_030761
4.31
GATA6
In utero embryonic development
NM_005257
4.17
TGFB1I1
Morphogenesis of embryonic epithelium
NM_001042454
4.13
TGFB3
In utero embryonic development
NM_003239
3.85
SOX5
In utero embryonic development
NM_152989
3.70
Developmental process
DES
Cytoskeleton organization
NM_001927
66.18
KRT6A
Ectoderm development
NM_005554
27.69
KRT5
Epidermis development
NM_000424
22.73
KRT13
Epidermis development, tongue morphogenesis
NM_153490
22.53
COL12A1
Skeletal system development, collagen fibril organization
NM_004370
17.87
DKK2
Multicellular organismal development
NM_014421
12.63
TAGLN
Muscle organ development
NM_001001522
11.91
KRT8
Cytoskeleton organization
NM_002273
11.04
KRT14
Epidermis development
NM_000526
9.01
OSTN
Ossification, multicellular organismal development
NM_198184
8.22
MCAM
Anatomical structure morphogenesis
NM_006500
7.22
IGF2BP3
Anatomical structure morphogenesis
NM_006547
7.10
ADAM19
Fertilization, muscle development, neurogenesis
NM_033274
6.41
MGP
Cartilage condensation, ossification
NM_001190839
5.70
DMD
Muscle organ development, skeletal muscle tissue development
NM_000109
5.22
PITX2
Multicellular organismal development
NM_153426
4.79
Physiological process
ACTG2
Muscle contraction
NM_001615
115.24
HBG1
Transport
NM_000559
101.65
PLN
Cellular calcium ion homeostasis, blood circulation
NM_002667
71.69
CNN1
Regulation of smooth muscle contraction
NM_001299
66.37
HBB
Regulation of blood pressure, oxygen transport
NM_000518
38.48
FGF10
Angiogenesis
NM_004465
31.24
COL8A1
Angiogenesis
NM_001850
12.02
AHSP
Hemoglobin metabolic process, hematopoiesis
NM_016633
7.18
FGF9
Angiogenesis, osteoblast differentiation
NM_002010
5.07
PDGFA
Angiogenesis, response to hypoxia
NM_002607
5.00
ELN
Respiratory gaseous exchange, blood circulation
NM_000501
4.75
EGF
Angiogenesis, positive regulation of cell proliferation
NM_001963
4.30
IL18
Angiogenesis, response to hypoxia
NM_001562
3.85
VEGFA
Angiogenesis, ovarian follicle development
NM_001025366
3.16
Signal transduction and regulation
PRLR
Cell surface receptor linked signaling pathway
NM_000949
90.61
RASSF3
Signal transduction
NM_178169
12.27
RERG
GTPase mediated signal transduction
NM_032918
11.98
STC2
Cell surface receptor linked signaling pathway
NM_003714
11.37
CD244
Signal transduction
NM_016382
8.83
ASPN
Negative regulation of transforming growth factor beta receptor signaling pathway
NM_017680
8.20
LPHN3
G-protein coupled receptor protein signaling pathway
NM_015236
7.72
SPSB1
Intracellular signaling pathway
NM_025106
6.87
ALCAM
Signal transduction, motor axon guidance
NM_001627
3.96
Cell regulation and proliferation
UPK1B
Epithelial cell differentiation
NM_006952
45.66
MYOCD
Cardiac cell differentiation
NM_001146312
40.32
EGFL6
Cell differentiation
NM_015507
11.94
PODN
Negative regulation of cell proliferation
NM_153703
9.96
FAS
Positive regulation of necrotic cell death
NM_000043
9.34
KRT4
Epithelial cell differentiation, negative regulation of epithelial cell proliferation
NM_002272
6.52
IGFBP7
Regulation of cell growth
NM_001553
6.01
HEMGN
Cell differentiation, regulation of osteoblast differentiation
NM_018437
5.70
CDH1
Trophectodermal cell differentiation
NM_004360
5.44
DPT
Cell adhesion, negative regulation of cell proliferation
NM_001937
5.13
Cytokine,  chemokine, and immune response
IL1RL1
Immune response
NM_016232
36.22
S100A8
Chemotaxis, inflammatory response
NM_002964
26.48
DPP4
Regulation of T cell mediated immunity
NM_001935
18.16
ITGB6
Inflammatory response
NM_000888
15.01
IGF2BP1
Regulation of cytokine biosynthetic process
NM_006546
9.59
LY96
Inflammatory response, cellular defense response
NM_015364
8.40
CCRL1
Chemotaxis, immune response
NM_178445
7.81
ANLN
Cytokinesis
NM_018685
7.60
CMKLR1
Chemotaxis, immune response
NM_001142343
7.41
CD97
Inflammatory response, immune response
NM_078481
6.96
CXCL1
Chemotaxis, inflammatory response, immune response
NM_001511
5.54
IL33
Positive regulation of macrophage activation
NM_033439
5.29
CXCR1
Chemotaxis, inflammatory response
NM_000634
3.48
CXCL6
Chemotaxis, inflammatory response
NM_002993
3.07Table 3
Representative differentially expressed genes with higher expression levels in dental pulp tissue than in umbilical cord.
Functional category
Gene symbol
Biological process
Accession number
Absolute fold change
Biomineral tissue development
PHEX
Bone and dentin mineralization
NM_000444
160.01
DMP1
Bone and dentin mineralization
NM_004407
99.20
CALB1
Hydroxyapatite formation
NM_004929
98.07
MMP20
Regulation of enamel mineralization, proteolysis
NM_004771
85.26
AMBN
Bone mineralization, odontogenesis of dentine-containing tooth
NM_016519
65.69
DSPP
Odontogenesis of dentine-containing tooth
NM_014208
41.26
ALPL
Biomineral tissue development
NM_000478
14.83
DLX1
Odontogenesis of dentine-containing tooth
NM_178120
9.55
LHX8
Odontogenesis of dentine-containing tooth
NM_001001933
7.56
CA2
Odontogenesis of dentine-containing tooth
NM_000067
7.07
RUNX2
Ossification, osteoblast differentiation
NM_001024630
6.53
LEF1
Odontogenesis of dentine-containing tooth
NM_016269
6.24
PAX9
Tooth development
NM_006194
5.79
MSX1
Odontogenesis, craniofacial development
NM_002448
4.04
WNT10A
Regulation of odontogenesis of dentine-containing tooth
NM_025216
3.87
Developmental process
DLX5
Osteoblast differentiation
NM_005221
30.01
DLX3
Multicellular organismal development
NM_005220
26.13
ADAM22
Proteolysis, central nervous system development
NM_021723
25.00
NES
Central nervous system development
NM_006617
15.67
BMP7
Ossification, organ morphogenesis
NM_001719
15.60
BMPR1B
Skeletal system development, cartilage condensation
NM_001203
13.53
MSX2
Skeletal system development, osteoblast differentiation
NM_002449
9.32
COL11A2
Skeletal system morphogenesis, cartilage development, palate development
NM_001163771
8.55
BMP5
Skeletal system development, ossification
NM_021073
6.08
TBX3
Blood vessel development, in utero embryonic development
NM_016569
5.93
MBP
Central nervous system development, myelination
NM_001025101
4.95
DLX6
Multicellular organismal development, nervous system development
NM_005222
4.12
NRCAM
Axonogenesis, central nervous system development
NM_001193582
4.09
SNAI1
Osteoblast differentiation, mesoderm formation
NM_005985
3.78
HLF
Multicellular organismal development, rhythmic process
NM_002126
3.23
BMP2
Development of bone and cartilage
NM_001200
2.11
Physiological process
TF
Controlling iron concentrations, erythropoiesis
NM_001063
153.98
SCN7A
Sodium ion transport, muscle contraction
NM_002976
82.36
APOD
Lipid metabolic process
NM_001647
41.22
PLAT
Response to hypoxia, blood coagulation
NM_000930
10.20
CD52
Elevation of cytosolic calcium ion concentration
NM_001803
4.29
CYGB
Response to oxidative stress, oxygen transport
NM_134268
3.98
MYOT
Muscle contraction
NM_006790
3.26
Signal transduction and regulation
WIF1
Wnt receptor signaling pathway
NM_007191
70.19
PTN
Transmembrane receptor protein signaling pathway
NM_002825
38.17
WNT5A
Oncogenesis, embryogenesis
NM_003392
13.68
NCAM1
Neurite outgrowth, synaptic plasticity
NM_181351
7.87
NCAM2
Neurite outgrowth, synaptic plasticity
NM_004540
7.38
RASGRF2
Induction of apoptosis by extracellular signals
NM_006909
6.88
CHN1
Signal transduction
NM_001822
6.56
MET
Cell surface receptor linked signaling pathway
NM_001127500
3.01
Cell regulation and proliferation
SCIN
Negative regulation of cell proliferation
NM_001112706
58.65
RELN
Cell morphogenesis involved in differentiation
NM_005045
49.12
NES
Structural organization of the cell
NM_006617
15.67
MEGF10
Phagocytosis
NM_032446
9.89
EPCAM
Positive regulation of cell proliferation
NM_002354
8.79
PDGFD
Positive regulation of cell division
NM_025208
5.43
CLU
Cell death, positive regulation of cell proliferation
NM_001831
5.19
VEGFC
Angiogenesis, positive regulation of neuroblast proliferation
NM_005429
3.74
MSI2
Stem cell development
NM_138962
3.55
DBC1
Cell cycle arrest, cell death
NM_014618
3.32
TGFB1
Cell growth, cell proliferation, cell differentiation and apoptosis
NM_000660
3.14
IGFBP6
Regulation of cell growth
NM_002178
3.07
Cytokine, chemokine, and immune response
IGJ
Immune response
NM_144646
22.65
IGHD
Immune response
BC021276
11.30
CXCL14
Chemotaxis, immune response, inhibiting angiogenesis
NM_004887
8.74
SELE
Leukocyte migration involved in inflammatory response
NM_000450
5.82
MX1
Induction of apoptosis, defense response, response to virus
NM_002462
5.24
IFI44
Response to virus
NM_006417
5.05
CX3CL1
Chemotaxis, defense response, immune response
NM_002996
3.60
CFI
Innate immune response
NM_000204
3.17


## 3.2. Gene Ontology Analysis

In the UC tissue, the expression levels of 158 genes were upregulated 10-fold or more in comparison with DP, whereas the expression levels of 118 genes were upregulated 10-fold in DP in comparison with UC. Genes related to cell proliferation and angiogenesis were expressed at higher levels in UC than in DP. Lipid metabolic process-related genes were highly expressed in DP. In comparison with DP, a large proportion of the UC genes were related to growth factor activity, structural molecular activity, DNA binding, and protein binding (Figure1).Figure 1
Main categories of genes expressed specifically in umbilical cord and dental pulp classified according to their biological (a) and molecular functions (b). Genes related to cell proliferation and angiogenesis were expressed at higher levels in the UC than in DP (p<0.05).
(a)
(b)


## 3.3. Stemness Characterization Using Surface Protein Markers

The comparative expression results for MSC surface protein markers are indicated in Figure3. UC tissue appeared to contain a population of cells that were more positive for MSC markers (includingCD29,CD34,CD44,CD73,CD105,CD146, andCD166) according to the minimal criteria of the International Society for Cell Therapy [18]. The comparative expression analysis of four induced pluripotent stem cell (iPSC) marker genes (i.e.,OCT4,SOX2,MYC, andKLF4) revealed that the expression level of these genes was the same in UC and DP. qRT-PCR analysis for eight important marker genes revealed four tooth-related genes (DSPP,AMBN,CALB1, andDMP1), three genes for MSCs (CD34,CD146, andCD166), and MYC related to iPSC stemness. DSPP, AMBN, CALB1, and DMP1 were expressed in DP tissue but they were not excluded in the comparative qPCR results because they were not detected in UC tissue. The expression levels of CD146, CD166, and MYC were 18.3, 8.24, and 1.63 times higher, respectively, in UC than in DP, and the expression level of CD34 was 2.15 times higher in DP than in UC (Figure 2).Figure 2
Relative gene expression of mesenchymal stem cell and induced pluripotent stem cell markers using cDNA microarray (a). The relative differences in the expression of stem cell markers between umbilical cord and dental pulp were analyzed using qRT-PCR (b). Data are presented as means ± standard deviation and expressed as the relative change by applying the equation2-ΔCt, where ΔCt = Ct of the gene minus Ct of the 18S rRNA.
(a)
(b)Figure 3
Immunohistochemical (IHC) staining of umbilical cord tissues. IHC staining for DSPP (a, e), DMP1 (b, f), CALB1 (c, g), and CD146 (d, h). CD146 is expressed on arteries of the umbilical cord, suggesting that the majority of stem cells arise from the microvasculature. (scale bars: (a)–(d) 4 mm, (e)–(h) 200μm).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.4. Immunohistochemical Staining

MCAM (CD146) was expressed abundantly as a perivascular stem cell marker in connective tissue, especially in the arteries of the UC. However, this protein showed a very low level of expression in DP tissue (Figure3). IHC staining showed that DSPP, DMP1, and CALB1 were expressed broadly in DP tissue (Figure 4), but barely expressed in the UC. DSPP was evident in pulpal tissue, the odontoblast layer, and primary and secondary dentin. While DMP was stained in the odontoblast layer and secondary dentin, CALB1 was expressed in pulpal tissue and the odontoblast layer. These findings were consistent with the microarray data.Figure 4
Immunohistochemical (IHC) staining of dental pulp tissue. IHC staining for DSPP (a, e). DSPP was noted in pulpal tissue, the odontoblast layer, and primary and secondary dentin. IHC staining for DMP1 (b, f) and CALB1 (c, g). CALB1 was especially expressed in pulpal tissue and the odontoblast layer. IHC staining for CD146 (d, h) (scale bars: (a)–(d) 4 mm, (e)–(h) 200μm).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 4. Discussion

Although numerous studies have been performed to evaluate the utility of human MSCs as sources for the development of cell-based therapeutics, a precise understanding of the biology of MSCs remains elusive. Previous studies, based on a strict definition of MSCs at the molecular or cellular levels, involved serial analyses of gene expression. However, such approaches raised concerns that the expression of housekeeping genes may have interfered with the identification of MSC-specific genetic characteristics. To overcome such shortcomings, we examined DNA microarray-based differential expression profiles of MSC population, comparing these profiles with those of other tissues to evaluate stemness capacity. We compared DP with UC tissue as both types contain stromal stem cell populations with high proliferative potential that are capable of regenerating their respective microenvironments with remarkable fidelity [19]. In order to identify genes expressed at higher levels in UC than in DP tissue, we categorized genes according to the ratio of the fold change between the two tissues. Results of qRT-PCR and IHC staining analyses confirmed the validity of these data, indicating that the expression of representative genes was consistent with the differential expression patterns observed in the microarray data.Although MSCs originating from both tissues are highly similar, their differences may be functionally related to their origin; for example, MSCs derived from DP are more committed to the osteoblastic and odontogenic lineages, whereas MSCs derived from the UC would be more committed to angiogenesis. In DP tissue, microarray results indicated that certain genes (DMP1,AMBN,DSPP,DLX1,RUNX2,LEF1,PAX9, andMSX1) related to odontogenesis and biomineral tissue development were upregulated, which was in agreement with the expected results for this biological process. In addition, genes that might be related to bone and dentin mineralization, includingPHEX,CALB1,MMP20,ALPL,LHX8, andWNT10A, were upregulated. Microarray data showed that the expression ofDMP1,DSPP, andCALB that play important roles in the development of pulp tissue was 99.2, 98.1, and 41.3 times higher, respectively, in DP than in UC. qRT-PCR results indicated that the fold differences in the expression ofDMP1,CALB1, andAMBN were not observed in the UC. Similarly, IHC staining results showed thatDMP1,CALB1, andDSPP were not stained in the UC but were stained around the outer area of DP. The genetic pattern analysis of permanent pulp indicated thatCALB1, a representative gene in DP, is necessary for enamel mineralization in transition- and maturation-stage ameloblasts [17].MSCs possess multilineage differentiation potential with a variety of chemokines, cytokines, and growth factors involved in the regeneration of damaged tissue. They are capable of modifying their molecular activities and functions in response to the environment. The exclusive expression of the chemokines CXCL1 and CXCL6 in the UC may increase propagation of hematopoietic precursors in coculture settings. Other genes expressed at higher levels in the UC include those encoding IL-6, IL-18, FGF9, FGF10, PDGFA, EGF, and VEGFA, which are part of interconnected pathways related to angiogenesis. Jin et al. reported that MSCs derived from bone marrow, adipose tissue, and the UC have significantly different anti-inflammatory capacities and confirmed that UC-MSCs exhibit the greatest anti-inflammatory effects [20]. These findings suggest that UC-MSCs are more efficient for clinical applications involving revascularization.In this study, the comparison of stemness of UC and DP tissues revealed no significant fold difference in the expression of several surface markers (CD29, CD34, CD44, CD73, CD105, and CD106) typical for MSCs. Nevertheless, some differences were observed in the expression level of CD146 (MCAM) and CD166 (ALCAM), which connect the control of cell growth with cell migration. These findings are representative of the developmental process. The qRT-PCR results showed that the expression levels of CD146 and CD166 were higher in UC than in DP (18.3-fold and 8.24-fold, resp.). These molecular differences in tissue-specific MSC gene expression may reflect their functional activities in distinct niches. A study utilizing flow cytometry reported higher expression of CD146, a marker expressed on both BMSCs and DP-MSCs [21]. IHC data confirmed that CD146 is a marker of vascular endothelial cells expressed on arteries of the UC and the outer walls of blood vessels in DP, suggesting that the majority of stem cells arise from the microvasculature. Accumulating evidence suggests that the expression of CD166 reflects the onset of a cellular program involving neural development, branching organ development, hematopoiesis, the immune response, and tumor progression [22]. Struys et al. reported that cultured DPSCs and UC-MSCs showed a similar expression pattern of antigens characteristic of MSCs such as CD105, CD29, CD44, CD146, and STRO-1 [23]. DPSCs are also identified by their positive expression of CD29, CD44, CD73, CD90, CD105, and STRO-1 [19]. CD34 protein is a specific antigen in hematopoietic cells, indicating that a greater number of immature hematopoietic cells are present in both UC and DP [24]. CD34 is present on the outer cell walls of DP and in the connective tissue of the UC, in agreement with previous studies reporting that CD34 localizes on large blood vessels, but not capillaries [25].The expression of pluripotent stem cell genes in the UC and DP might reflect their embryonic origin. iPSCs are the most promising cell source for cell-based therapy in regenerative medicine, as they give rise to development by introducing 4 factors: MYC, KLF4, OCT4, and SOX2 [7]. No significant differences were found between the expressions of these factors in the two tissue types; MYC, KLF4, OCT4, and SOX2 were expressed 1.57, 1.03, 1.20, and 1.20 times more highly, respectively, in UC than in DP tissue. Previously, DPSCs were characterized by the low levels of expression of undifferentiated cell-associated genes, such as OCT4, MYC, and Nanog, which are considered to facilitate reprogramming [26]. Recent studies utilizing the UC to derive iPSCs are expected to contribute to the further expansion of its pluripotency for therapeutic purposes, including drug discovery [27, 28].The UC expresses specific embryonic cell markers such as DLK1, DKK1, TBX18, WNT4, and TGFB3. Previous studies describing the expression of embryonic cell markers in the UC have shown that fetal perivascular cells express Runx1 and OCT-4 at different levels, which characterizes the undifferentiated stem cell state [29]. Interestingly, UC-MSCs exhibited higher levels of expression of genes related to cell proliferation than DPSCs, whereas DPSCs exhibited a higher proliferation rate compared with BMSCs in vitro [11]. Previous study revealed similar result of this research that UC-MSCs seemed to have higher cell proliferation ability, while DP-MSCs may have significant differences for lower cell apoptosis, osteogenic differentiation, and senescence [30]. This may be attributed to the developmental state of tissues, as UC samples are at an earlier stage of development compared with DP from fully developed and erupted permanent teeth. Commonly expressed genes in DP include those coding for various growth factors (BMP-2, BMP-5, BMP-7, MMP20, and TGF-β1) implicated as strong promoters of the formation of mineralized bone matrix and tooth morphogenesis [31]. Coexpression of genes with known functions, and unknown or novel genes, may provide a simple means to obtain data about genes for which little information is available.Although the results of this study are still limited and require further investigation using additional methods, the similarity between MSCs derived from the UC and DP at the transcription level definitively places both tissues as potentially more accessible sources of MSCs. Furthermore, the present gene expression analysis confirms similarities between MSCs derived from the UC and DP and provides molecular and biological insights into the developmental mechanisms involved in angiogenic and odontogenic processes.


## 5. Conclusions

Here, we presented comparative gene expression data for human UC and DP tissue. Although UC tissue showed similar but slightly higher expression patterns, with the usual MSC markers, both tissues clearly diverged in their differentiation capacity. Further research is necessary to understand and describe the significance of these findings for clinical applications.



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